Blood-brain barrier transmigrating compounds and uses thereof

ABSTRACT

A brain-penetrating composition of amyloid-ß binding peptide is disclosed. This may be useful in the treatment of Alzheimer&#39;s disease, for example as a bifunctional molecule, comprising a blood-brain barrier crossing antibody and an amyloid-ß targeting peptide linked via an Fc fragment that is able to transmigrate across the blood-brain barrier into the brain, and compositions comprising same. Methods of using this composition for treating Alzheimer&#39;s disease are disclosed.

FIELD OF THE INVENTION

The present invention relates to compounds that transmigrate the blood-brain barrier, and uses thereof. More specifically, the present invention relates to compounds that may comprise an antibody or fragment thereof that crosses the blood-brain barrier, an immunoglobulin Fc domain or fragment thereof, and a polypeptide binding to beta-amyloid, fusion proteins and compositions thereof and their use in the treatment of Alzheimer's disease.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 of international application number PCT/IB2018/055747, filed Jul. 31, 2018, which claims the benefit of international application number PCT/IB2018/050576, filed Jan. 30, 2018, each of which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, are an increasing burden on our ageing society because there are currently no effective treatments for these disabling conditions. Alzheimer's disease (AD) is an irreversible neurodegenerative disorder affecting approximately 15% of the population over 65 years of age and is the predominant cause of progressive intellectual and cognitive failure in the ageing population (Hardy et al, 2014).

In AD, there is a severe loss of cholinergic neurons with a consequent decline in the levels of acetylcholine (ACh), a key neurotransmitter involved in memory processing and storage. In addition, excitotoxicity induced by neurotransmitter glutamate is also implicated in the pathogenesis of AD. Therefore, cholinergic augmentation and/or inhibition of glutamate toxicity might improve cognition in AD. Indeed, the only FDA approved drugs for the treatment of AD are acetylcholine esterase (AChE) inhibitors (e.g., donepezil, rivastigmine, galantamine) to prevent the loss of ACh and inhibitors of specific glutamate receptors (e.g., memantine) (Mangialasche et al, 2010; Ji and Ha, 2010; Savonenko et al, 2012). However, the beneficial effects of these symptomatic drugs are limited and transient, providing temporary improvement in cognitive functions and do not stop the progression of the disease. While other treatments including antioxidants, anti-inflammatory drugs (NSAIDS), cholesterol-lowering drugs and estrogen therapy are considered, none of these treatments appear to have any long-term beneficial effects, particularly in improving memory and cognitive function in AD patients (Magialasche et al, 2010; Ji and Ha, 2010).

A major hallmark of Alzheimer's disease is the accumulation of a 39-43 amino acid peptide β-amyloid (Aβ) in the brain in the form of aggregates and plaques. A considerable body of evidence based on genetic, pathological and biochemical studies indicate that Aβ, particularly its oligomeric aggregates, plays a central role in the development of AD pathology (Hardy et al, 2014; DeLaGarza, 2003; Selkoe and Hardy, 2016). According to amyloid hypothesis, a chronic imbalance in the production and clearance of Aβ in the brain results in its accumulation and aggregation with ageing. These Aβ aggregates are believed to initiate a cascade of events leading to synaptic loss and neuronal functions, leading to a progressive loss of memory and other cognitive functions (Hardy et al, 2014; DeLaGarza, 2003; Selkoe and Hardy, 2016; Sengupta et al, 2016).

The generation of Aβ from its precursor protein APP is achieved by the sequential proteolysis of APP by proteases β and γ secretases (Barageb and Sonawane, 2015). Inhibitors of these enzymes have been shown to reduce Aβ production and are being developed as potential drugs for treating AD (Hardy et al, 2014; Mangialasche et al, 2010; Selkoe and Hardy, 2016; Ji and Ha, 2010). Similarly, agents that sequester and/or promote Aβ clearance are also being developed. Notable among these are the development of immunotherapies with AD vaccine. Both active (Aβ peptides) and passive immunization (Aβ-antibodies) have been shown to be effective in preventing amyloid deposition as well as clearing of preformed amyloid plaques in transgenic animal models of AD and in clinical trials involving AD patients (Mangialasche et al, 2010; Ji and Ha, 2010; Morrone et al, 2015; Lannfelt et al, 2014; Selkoe and Hardy, 2016; Goure et al, 2014).

Inhibitors of β and γ secretases that prevent proteolytic cleavage of amyloid precursor protein (APP) and thereby reduce or suppress brain Aβ production are being developed (e.g., tarenflurbil, semagacestat, verubecestat). However, their therapeutic efficacy in reducing Aβ burden is not yet known and many of these drugs have failed in pre-clinical or clinical trials (Savonenko et al, 2012; Musiek and Holtzman, 2015). Moreover, since these enzymes are also involved in the processing of other enzymes and signaling molecules such as Notch that are linked to neuronal development (Savonenko et al, 2012; Musiek and Holtzman, 2015), these inhibitors may have serious non-specific side effects.

Immunotherapeutic approaches such as active (Aß vaccine, AN1792) and passive immunization (e.g., Bapineuzumab, Solanezumab, Crenezumab, aducanumab etc) have been shown to be quite effective in reducing Aβ deposition and partial elimination of memory deficits in transgenic animals (Monsonego and Weiner, 2003; Bard et al, 2000, Sevigny J et al., 2016). Several clinical trials using both active and passive immunization have shown reduction in brain Aβ deposition with moderate improvement in cognition. However, clinical trials had to be abandoned due to severe inflammatory reactions (meningo-encephalitic presentation), vasogenic edema, and micro-haemorrhages in AD patients. Despite these limitations, the immunotherapy approach indicates that agents that effectively sequester Aβ, and prevent its deposition and toxicity, could potentially serve as effective drugs in arresting the progression of AD, and even prevent its development (Rafii and Aisen, 2015, Selkoe and Hardy, 2016).

Treatment as well as early diagnosis of AD and other diseases that originate in the brain remain challenging because the majority of suitable therapeutic molecules and diagnostics cannot penetrate the tight and highly restrictive blood-brain barrier (BBB) (Abbott, 2013). The BBB constitutes a physical barricade that is formed by brain endothelial cells (BECs) that line the blood vessels and connect with each other through tight junctions (Abbott, 2013). The tight junctions formed between the BECs are essential for the integrity of the BBB and prevent the paracellular transport of molecules larger than 500 daltons (Da). Because brain endothelial cells exhibit very low pinocytosis rates (Abbott, 2013), transcellular transport of larger molecules is limited to the highly specific receptor mediated transcytosis (RMT) pathway, and the passive, charge-based adsorption mediated transcytosis (Abbott, 2013; Pardridge, 2002). Additionally, the high density of efflux pumps, such as P-glycoprotein or the multi-drug resistance protein-1 (MDR-1), contribute to the removal of unwanted substances from the brain (Abbott, 2013).

While all these characteristics protect the brain from pathogens and toxins, they equally prevent the entry of most therapeutics. In fact, less than 5% of small molecule therapeutics and virtually none of the larger therapeutics can cross the BBB in pharmacologically relevant concentrations (i.e., sufficient to engage a central nervous system (CNS) target and elicit pharmacologic/therapeutic response) unless they are specifically ‘ferried’, that is, coupled to a transporter molecule. Due to the lack of effective ‘carriers’ to transport molecules across the BBB, numerous drugs against neurodegenerative diseases have been ‘shelved’ or eliminated from further development as they cannot be delivered to the brain in sufficient amount.

Despite considerable progress in understanding the molecular mechanism of AD pathology, there are no effective drugs or treatments currently available that can prevent the progression of or cure the disease. Furthermore, the lack of high-capacity and high-selectivity BBB carriers delays the development of new therapeutics and diagnostics for diseases originating in the brain, including brain tumors and neurodegenerative diseases.

SUMMARY OF THE INVENTION

The present invention relates to compounds or compositions that transmigrate the blood-brain barrier, and uses thereof.

The present invention provides peptides which bind beta-amyloid (ß-amyloid). The peptides (or proteins) that bind β-amyloid may selectively bind pathologically relevant ß-amyloid₁₋₄₂ (Aβ₁₋₄₂) aggregates, and may be abbreviated and referred to herein as ABP, wherein ABP herein encompasses any ABP peptide, ABP variant, any C-terminally cleaved ABP, or equivalent functional β-amyloid binding peptide thereof. The present invention provides specific ABP peptide species that bind ß-amyloid, and advantageously provides a mixture of functional ß-amyloid binding peptides, and accordingly a mixture of stable and functional fusion proteins comprising said specific ABP peptides.

The present invention additionally provides fusion proteins (also referred to herein as compounds, compositions, single-chain polypeptides or constructs) comprising a beta-amyloid (ß-amyloid) binding peptide (any ABP provided herein) linked to an antibody or fragment thereof that crosses the blood-brain barrier (BBB), wherein BBB herein refers to an abbreviation for the carrier antibody or fragment that transmigrates the blood brain barrier. In an embodiment, the fusion protein comprising an ABP and a BBB carrier, or fragment thereof, wherein the ABP and BBB components of the fusion protein may be linked via an Fc region or portion thereof. In an embodiment, the fusion protein comprising an ABP and a BBB, further comprises an immunoglobulin protein effector domain, known as Fc, or fragment thereof, wherein the ABP and BBB components of the fusion protein may be linked via the Fc region or portion thereof. For example, a construct of the present invention may further comprise a linker (L), wherein L is a small linking peptide or peptide-like chain. The single chain polypeptide comprising BBB-Fc-ABP may form a dimer through Fc which allows the dimerization of the fusion protein. A compound of the present invention may be referred to as a fusion protein, construct, fusion molecule, formulation or composition.

The single chain polypeptide comprising a BBB-Fc-L-ABP fusion protein may form a dimer by Fc dimerization, wherein the BBB-Fc-L-ABP may be a homodimer or heterodimer with respect to ABP. That is to say, the BBB-Fc-L-ABP fusion protein provided may be a single chain polypeptide or a dimeric polypeptide thereof, the dimer may be a homodimer in which both the ABP arms are functional (ABP and/or C-terminally cleaved functional ABP), or the dimer may be a heterodimer comprising a functional ABP arm (ABP or C-terminally cleaved functional ABP) and a non-functional ABP arm, or two functional ABP arms. The term “functional” indicates that the ABP peptide is capable of binding β-amyloid, such as a peptide having consensus sequence of SEQ ID NO: 31 (40 aa) and C-terminal cleaved functional products thereof, or having a consensus sequence of SEQ ID NO: 46 (29 aa); while the term ‘non-functional’ indicates any C-terminal cleavage product that does not allow for the ABP to bind β-amyloid. Fc dimerization may be mediated by interaction of a large tightly packed hydrophobic interface between two Fc CH3 domains in a construct comprising BBB-Fc-ABP. It should be noted that herein BBB-Fc-ABP and BBB-Fc-L-ABP are equivalent abbreviations referring to a single-chain polypeptide fusion protein comprising a BBB, an Fc portion, and an ABP, wherein each component of the fusion protein is linked or conjugated via any suitable linker to form a β-amyloid binding fusion protein capable of transmigrating the blood brain barrier. The BBB, Fc, ABP and linkers (L) may be any BBB, Fc, ABP or L provided herein. In an embodiment, BBB is FC5 (for example SEQ ID NO: 17), Fc is hFc1X7 (for example, SEQ ID NO:49), ABP is a β-amyloid binding peptide, such as any peptide having SEQ ID NO: 31 (40 aa), for example ABP(6G) SEQ ID NO: 36 (40 aa) or any β-amyloid binding C-terminally cleaved product thereof, for example SEQ ID NO: 37 (38 aa), SEQ ID NO: 38 (37 aa), SEQ ID NO: 39 (36 aa), SEQ ID NO: 40 (35 aa), SEQ ID NO: 41 (34 aa), SEQ ID NO: 42 (33 aa), or SEQ ID NO: 43 (32 aa) or any peptide comprising or consisting of SEQ ID NO: 46 (29 aa), for example SEQ ID NO: 47 (29 aa); and L may be (GGGGS)n, (GGGS)n or any suitable linker thereof, for example, T(GGGGS)₂. It is understood that the provided fusion protein is not limited to a specific linker and may be any linker that allows for the operable biological function of each component of the fusion protein, namely blood-brain barrier transmigration and β-amyloid binding. For example, a fusion protein of the present invention may be FC5-H3-hFc1X7-L-ABP(6G) as provided in SEQ ID NO: 56, 71 or any equivalent fusion protein thereof, such as fusion protein comprising any β-amyloid binding protein provided herein or a C-terminally cleaved product thereof.

An equivalent fusion protein thereof may comprise any β-amyloid binding protein (SEQ ID NO: 31), a C-terminally cleaved product of SEQ ID NO: 36 (eg. SEQ ID NO: 37 to SEQ ID NO 43), or a protein comprising or consisting of SEQ ID NO: 46, or an equivalent β-amyloid binding protein thereof. For example, a C-terminally cleaved ABP may comprise a sequence of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.

The BBB-Fc-ABP construct provided may be a single chain polypeptide (referred to as a monomer of the single chain fusion protein) or a dimeric polypeptide thereof. The dimeric peptide comprising two single chain polypeptides may be a homodimer—both ABP peptides are the same (e.g., both SEQ ID NO: 36); or the dimeric peptide may be a heterodimer (eg. SEQ ID NO: 36 and SEQ ID NO: 40 or any combination of SEQ ID NO: 31 to 47)—the ABP peptides of the dimer may be any two different sequences wherein both or at least one of the APB peptides of the single chain is selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43. The ABP peptide of the single chain polypeptide forming a dimer may be any biologically functional β-amyloid binding ABP peptide; specifically any ABP selected from the group consisting of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 44, 45, 46, 47, or a C-terminally cleaved biologically functional SEQ ID NO: 36 product, specifically, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43 or any combination thereof.

The dimeric peptide comprising two BBB-Fc-L-ABP single chain polypeptides may comprise two different ABP sequences, wherein at least one of the said ABP sequences is a biologically functional molecule (i.e. can bind β-amyloid). It is quite advantageous and unexpected that the dimeric peptide may accommodate a single non-functional ABP peptide and still maintain an equivalent functional biological activity with respect to β-amyloid binding. In a dimer comprising two different ABP sequences (i.e. heterodimeric with respect to ABP), at least one of the ABP peptides provided in the heterodimer be a biologically functional ABP i.e. SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 44, 45, 46, 47 or any combination thereof when two functional ABP peptides are provided in the heterodimer. It is not necessary that both ABP peptides in the heterodimer be biologically functional β-amyloid binding proteins, having at least one biologically functional ABP in the heterodimer maintains the overall biological activity of the fusion protein with respect to β-amyloid binding activity. It is advantageous that a heterodimer can accommodate a single non-functional ABP peptide and remain a biologically effective heterodimer. This advantage aids in the manufacturability of the provided fusion protein and allows significantly improved yield in a mixture of biologically active product.

The present invention provides an isolated peptide that binds β-amyloid comprising or consisting of an amino acid sequence of:

X₁TFX₂TX₃X₄ASAQASLASKDKTPKSKSKKX₅X₆

-   -   where X₁=K, G or A, X₁=G or A, X₂=K, G or V, X₃=R, G or A, X₄=K,         G or A, X₅=R, G or V, X₆=N, G or V, (SEQ ID NO: 46; 29 aa).

The present invention provides an isolated peptide that binds β-amyloid comprising or consisting of an amino acid sequence of:

(SEQ ID NO: 31; 40 aa) X₁TFX₂TX₃X₄ASAQASLASKDKTPKSKSKKX₅X₆STQLX₇SX₈VX₉NI

-   -   wherein X₁=K, G or A, X₁=G or A, X₂=K, G or V, X₃=R, G or A,         X₄=K, G or A, X₅=R, G or V, X₆=N, G or V, X₇=K, G or V, X₈=R, G         or A, X₉=K, G or A, or any C-terminally cleaved β-amyloid         binding product thereof.

The isolated peptide amino acid sequence comprises a sequence selected from the group consisting of:

(SEQ ID NO: 32; 40 aa) KTFKTRKASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI; (SEQ ID NO: 33; 40 aa) KTFKTRKASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI; (SEQ ID NO: 34; 40 aa) KTFKTRGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI; (SEQ ID NO: 35; 40 aa) KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI; (SEQ ID NO: 36); 40 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI; (SEQ ID NO: 37; 38 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVK; (SEQ ID NO: 38; 37 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV; (SEQ ID NO: 39; 36 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSR; (SEQ ID NO: 40; 35 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS; (SEQ ID NO: 41; 34 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLK; (SEQ ID NO: 42; 33 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQL; (SEQ ID NO: 43; 32 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQ; (SEQ ID NO: 44; 35 aa) KTFKTRKASAQASLASKDKTPKSKSKKGGSTVKNI; (SEQ ID NO: 45; 29 aa) KTFKTRKASAQASLASKDKTPKSKSKKRG; (SEQ ID NO: 47; 29 aa) GTFGTGGASAQASLASKDKTPKSKSKKGG; and a sequence substantially equivalent thereto, or a C-terminally cleaved β-amyloid binding peptide thereof. A C-terminal cleavage of one amino acid (i.e. the deletion of isoleucine) to yield a 39 amino acid cleavage product is also provided. In consensus sequence SEQ ID NO: 31, X₁₀=I or no amino acid (i.e. amino acid X₁₀ is deleted from SEQ ID NO: 30), wherein an —OH group may be present at the C-terminus of a 39aa peptide instead of an amino acid 40. (SEQ ID NO: 31)

More specifically, the C-terminally cleaved β-amyloid binding peptide comprises a sequence selected from the group consisting of:

(SEQ ID NO: 37; 38 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVK; (SEQ ID NO: 38; 37 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV; (SEQ ID NO: 39; 36 aa ) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSR; (SEQ ID NO: 40; 35 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS; (SEQ ID NO: 41; 34 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLK; (SEQ ID NO: 42; 33 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQL; and (SEQ ID NO: 43; 32 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQ.

Any isolated peptide of the present invention may be fused to an antibody or antibody fragment capable of transmigrating the blood brain barrier. The isolated peptide may be linked to an Fc fragment, wherein the isolated peptide, the antibody or fragment thereof, and the Fc fragment form a single-chain polypeptide fusion protein. In an embodiment, Fc fragment comprises an Fc with attenuated effector functions.

The present invention accordingly provides fusion protein comprising any isolated peptide provided herein, and an antibody or antibody fragment that transmigrates the blood brain barrier (BBB). This fusion protein may be a single-chain polypeptide further comprising an Fc fragment, wherein the single-chain polypeptide forms a dimer. The dimer may be a homodimer or a heterodimer with respect to the ABP comprised therein, and the heterodimer may be bifunctional (two functional ABP arms) or monofunctional (only one functional ABP arm). It is quite advantageous that the monofunctional dimers are equivalent to the bifunctional dimers, regardless of the functional ABP protein comprised therein. That is to say, for example a heterodimer can comprise an ABP having SEQ ID NO: 37 on one arm of the dimer, and SEQ ID NO: 41 on another arm of the dimer, or can comprise an ABP having SEQ ID NO: 43 on one arm and a non-functional ABP on the other arm of the dimer and both dimers advantageously exhibit equivalent β-amyloid binding functionality. This advantageously allows for the production of a mixture of dimers having equivalent functionality, which accordingly allows for an increased product yield.

Accordingly, there is provided fusion proteins wherein the dimer is a homodimer or heterodimer with respect to ABP, and comprises at least one ABP capable of binding β-amyloid.

The single-chain polypeptide comprises provided may comprise an antibody or antibody fragment thereof, a peptide that binds β-amyloid, and an Fc fragment. The single-chain polypeptide may further comprise any suitable linker that allows for the linking of the peptide that binds β-amyloid to the Fc fragment. The antibody or fragment thereof may be conjugated or linked to the Fc, and the ABP may be linked to the Fc through any suitable linker to form a single-chain polypeptide. The provided fusion proteins are not limited to comprise a linker as provided herein, and may include any suitable linker that allows for the linking of the components (namely the antibody, Fc, and ABP) to form a fusion protein equivalent to the fusion proteins provided herein.

The antibody or fragment thereof may be any antibody selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO: 25, and SEQ ID NO:26; the peptide that binds β-amyloid selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47; and the Fc fragment selected from the group consisting of SEQ ID NO: SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50.

The antibody or fragment thereof may comprise a sequence comprising a complementarity determining region (CDR) 1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGDNTFYSNSVKG (SEQ ID NO:2), a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:3). Specifically, the antibody or fragment thereof comprises a sequence selected from any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and a sequence substantially identical to any of the above sequences.

The antibody or fragment thereof may comprises a sequence selected from the group consisting of:

-   -   an antibody or fragment thereof comprising CDR1 sequence of         EYPSNFYA (SEQ ID NO:4), a CDR2 sequence of VSRDGLTT (SEQ ID         NO:5), a CDR3 sequence of AIVITGVWNKVDVNSRSYHY (SEQ ID NO:6);     -   an antibody or fragment thereof comprising CDR1 sequence of         GGTVSPTA (SEQ ID NO:7), a CDR2 sequence of ITWSRGTT (SEQ ID         NO:8), a CDR3 sequence of AASTFLRILPEESAYTY (SEQ ID NO:9); and     -   an antibody or fragment thereof comprising CDR1 sequence of         GRTIDNYA (SEQ ID NO:10), a CDR2 sequence of IDWGDGGX; where X is         A or T (SEQ ID NO:11), a CDR3 sequence of AMARQSRVNLDVARYDY (SEQ         ID NO:12).

Specifically, the antibody or fragment thereof may comprise a sequence selected from any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and/or a sequence substantially identical thereto.

The antibody or fragment thereof may be humanized. The antibody or fragment thereof may be a single-domain antibody (sdAb).

The fusion protein single-chain polypeptide comprising the antibody or fragment thereof is linked to the peptide that binds β-amyloid via the Fc fragment. The single-chain polypeptide may comprise any sequence selected from the group consisting of: SEQ ID NO:51 SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO: 54, SEQ ID NO:55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, and a sequence substantially identical thereto. The single-chain polypeptide may form a dimer, specifically a homodimer comprising any single-chain polypeptide provided or a heterodimer comprising at least one of the single-chain polypeptides provided.

The fusion protein may be a single-chain polypeptide comprising any suitable peptide linker. The peptide linker may comprise any amino acid sequence that allows for the linked components of the fusion protein to maintain their unrestricted desired biological function. For example, the peptide linker may comprise a sequence of (GGGS)n, (GGGGS)n, or any suitable peptide linking sequence.

In an embodiment, the single-chain polypeptides provided form a dimeric polypeptide, wherein the dimeric polypeptide comprises a homodimer or heterodimer with respect to the β-amyloid binding protein (ABP) comprised therein. The heterodimer may comprise at least one functional ABP capable of binding β-amyloid.

The Fc fragment of the fusion protein may be a mouse Fc2a or human Fc1; and may comprise an Fc fragment comprising SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50.

In an embodiment, the fusion protein comprises the antibody or fragment thereof linked to the N-terminus of the Fc fragment, and the polypeptide that binds β-amyloid is linked to the C-terminus of the Fc fragment; wherein the antibody or fragment is any antibody provided herein, for example, an antibody or fragment comprising CDRs of SEQ ID NO: 1, 2, 3 (FC5); CDRs of SEQ ID NO: 4, 5, 6 (IGF1R-3), CDRs of SEQ ID NO: 7, 8, 9 (IGF1R-4); CDRs of SEQ ID NO: 10, 11, 12 (IGF1R-5); SEQ ID NO: 14 to SEQ ID NO: 17 (FC5 antibodies); or SEQ ID NO: 18 to SEQ ID NO: 26 (IGF1R antibodies).

In another embodiment, the fusion protein comprises the antibody or fragment thereof linked to the C-terminus of the Fc fragment and the polypeptide that binds β-amyloid linked to the N-terminus of the Fc fragment; wherein the antibody or fragment is any antibody or fragment comprising; CDRs of SEQ ID NO: 4, 5, 6 (IGF1R-3), CDRs of SEQ ID NO: 7, 8, 9 (IGF1R-4); CDRs of SEQ ID NO: 10, 11, 12 (IGF1R-5); or SEQ ID NO: 18 to SEQ ID NO: 26 (IGF1R antibodies).

The fusion protein may comprise a linker, wherein the linker is an independently selected linker sequence linking the antibody or fragment thereof and/or linking the polypeptide that binds β-amyloid to the Fc. The linker may comprise the sequence (GGGS)n, (GGGGS)n, GGGGSGGGGS, GGGSGGGGS, any suitable linker as provided in the art, or as provided in the consensus linker of SEQ ID NO: 71 or any suitable linker.

The present invention provides dimers of any fusion protein provided, wherein the dimer comprises at least one peptide according to any one of SEQ ID NO: 31 to SEQ ID NO: 47, or any combination thereof.

More specifically, the fusion protein dimers comprise two single-chain polypeptides, wherein the two single-chain polypeptides are: bifunctional homodimers comprising two ABP selected from any one of SEQ ID NO: 31 to 47; bifunctional heterodimers comprising two different ABP selected from any one of SEQ ID NO: 31 to 47; or monofunctional heterodimers comprising one ABP selected from any one of SEQ ID NO: 31 to 47.

There is also provided a pharmaceutical composition comprising an isolated peptide of any one of SEQ ID NO: 31 to 47, or any combination thereof, and a pharmacologically acceptable carrier. The pharmaceutical composition may be used for treating Alzheimer's disease in a patient.

There is also provided a nucleic acid molecule encoding any protein or fusion protein of the present invention. There is provided a vector comprising the nucleic acid molecule encoding any protein or fusion protein of the present invention. There is provided a kit comprising the pharmaceutical composition.

The present invention provides a method of treating Alzheimer's diseases, comprising administering a pharmaceutical composition comprising any fusion protein, or dimer thereof to a subject in need thereof.

There is provided a method of reducing toxic ß-amyloid levels in the brain of a subject having increased levels of brain amyloid beta comprising the steps of repeated parenteral administration of a sufficient amount of a pharmaceutical composition provided to a subject.

The parenteral administration may be subcutaneous or intravenous administration. Reduction of ß-amyloid levels is not limited to the brain and can be in the brain, tissues, or biofluids (CSF and blood) of subjects having increased levels of ß-amyloid.

The provided method wherein toxic ß-amyloid levels are reduced, after repeated parenteral administration of the compositions provided in the brains of subjects having increased brain levels of amyloid beta. Toxic ß-amyloid are reduced within four weeks of repeated parenteral administration of the composition. Toxic ß-amyloid levels in the cerebrospinal fluid (CSF) of subjects having increased CNS (central nervous system) levels of ß-amyloid is reduced after parenteral administration of the composition. The provided method and compositions thereof advantageously reduced toxic ß-amyloid levels in the cerebrospinal fluid (CSF) in subjects having increased CNS levels within 24 hours of a single parenteral administration of the composition. Specifically toxic ß-amyloid levels were reduced in the brains of subjects having increased levels of brain amyloid beta in a method comprising the administration of the provided fusion proteins.

The pharmaceutical composition may comprise any mixture of fusion proteins provided; wherein the mixture of fusion proteins comprises ABP bifunctional homodimeric fusion proteins, ABP bifunctional heterodimeric fusion proteins and ABP monofunctional heterodimeric fusion proteins or any combination thereof. The pharmaceutical comprising a mixture of single-chain polypeptide fusions provides a pharmaceutically effective yield of functional fusion protein having equivalent functional activity. The pharmaceutical composition may comprise a pharmaceutically acceptable diluent, carrier, vehicle or excipient for ameliorating the symptoms of Alzheimer's disease. The pharmaceutical composition may be used to reduce toxic ß-amyloid levels in the brains of subjects having increased levels of brain amyloid beta.

The compounds of the present invention may be used in the treatment of Alzheimer's disease (AD).

The peptide that binds β-amyloid may comprise a sequence selected from the group consisting of:

(SEQ ID NO: 27; 174 aa) SGKTEYMAFPKPFESSSSIGAEKPRNKKLPEEEVES SRTPWLYEQEGEVEKPFIKTGFSVSVEKSTSSNRKN QLDTNGRRRQFDEESLESFSSMPDPVDPTTVTKTFK TRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKNI THARRILQQSNRNACNEAPETGSDFSMFEA (SEQ ID NO: 28; 40 aa) FSSMPDPVDPTTVTKTFKTRKASAQASLASKDKTPKSKSK; (SEQ ID NO: 29; 40 aa) KDKTPKSKSKKRNSTQLKSRVKNITHARRILQQSNRNACN; (SEQ ID NO: 30; 40 aa) KTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKNI;

There is provided a peptide that binds β-amyloid comprising a sequence:

X₁TFX₂TX₃X₄ASAQASLASKDKTPKSKSK KX₅X₆STQLX₇SX₈VX₉NI

-   -   where X₁=K, G or A, X₁=G or A, X₂=K, G or V, X₃=R, G or A, X₄=K,         G or A, X₅=R, G or V, X₆=N, G or V, X₇=K, G or V, X₈=R, G or A,         X₉=K, G or A (SEQ ID NO: 31; 40 aa), or a C-terminal cleavage         protein thereof [SEQ ID NO: 37 (38 aa) to SEQ ID NO: 43 (32         aa)].

In the consensus sequence of SEQ ID NO: 31, there is provided also variations in the substitutions at the variable amino acids; wherein X₁=G or A; X₂=G or V; X₃=G or A, X₄=G or A, X₅=G or V; or any combination of these amino acid substitutions in SEQ ID NO: 31.

There is also provided a peptide that binds β-amyloid protein comprising a sequence:

X₁TFX₂TX₃X₄ASAQASLASKDKTPKSKSKKX₅X₆

-   -   here X₁=K, G or A, X₁=G or A, X₂=K, G or V, X₃=R, G or A, X₄=K,         G or A, X₅=R, G or V, X₆=N, G or V, (SEQ ID NO: 46; 29 aa)

In the consensus sequence of SEQ ID NO: 46, there is provided also variations in the substitutions at the variable amino acids; wherein X₁=G or A; X₂=G or V; X₃=G or A, X₄=G or A, X₅=G or V; or any combination of these amino acid substitutions in SEQ ID NO: 46.

X In specific non-limiting embodiments, the ABP, its variants, or C-terminal products thereof may comprise a sequence selected from any one of:

X₁TFX₂TX₃X₄ASAQASLASKDKTPKSKSKKX₅X₆ STQLX₇SX₈VX₉NI

-   -   where X₁=K, G or A, X₁=G or A, X₂=K, G or V, X₃=R, G or A, X₄=K,         G or A, X₅=R, G or V, X₆=N, G or V, X₇=K, G or V, X₈=R, G or A,         X₉=K, G or A (SEQ ID NO:31; 40 aa),

(SEQ ID NO: 32; 40 aa) KTFKTRKASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI; (SEQ ID NO: 33; 40 aa) KTFKTRKASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI; (SEQ ID NO: 34; 40 aa) KTFKTRGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI; (SEQ ID NO: 35; 40 aa) KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI; (SEQ ID NO: 36; 40 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI; a C-terminal cleavage product of SEQ ID NO: 36, wherein one amino acid (Isoleucine) is deleted from the C-terminus.

(SEQ ID NO: 37; 38 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVK; (SEQ ID NO: 38; 37 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV; (SEQ ID NO: 39; 36 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSR; (SEQ ID NO: 40; 35 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS; (SEQ ID NO: 41; 34 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLK; (SEQ ID NO: 42; 33 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQL; (SEQ ID NO: 43; 32 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQ; (SEQ ID NO: 44; 35 aa) KTFKTRKASAQASLASKDKTPKSKSKKGGSTVKNI; (SEQ ID NO: 45; 29 aa) KTFKTRKASAQASLASKDKTPKSKSKKRG; X₁TFX₂TX₃X₄ASAQASLASKDKTPKSKSKKX₅X₆

-   -   where X₁=K, G or A, X₁=G or A, X₂=K, G or V, X₃=R, G or A, X₄=K,         G or A, X₅=R, G or V, X₆=N, G or V, (SEQ ID NO: 46; 29 aa); and

(SEQ ID NO: 47; 29 aa) GTFGTGGASAQASLASKDKTPKSKSKKGG; or a sequence substantially identical to any of the above sequences, capable of binding β-amyloid.

The ABP that binds β-amyloid may comprise a peptide sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43. SEQ ID NO: 44, and SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 An ABP comprising or consisting of a peptide sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, may be referred to as an ABP, an ABP variant, a C-terminal cleavage product or any equivalent β-amyloid binding peptide. The present invention additionally comprises an ABP sequence having consensus sequence SEQ ID NO: 31 or a C-terminally functional product thereof, or a consensus sequence of SEQ ID NO: 46; for example, ABP may comprise sequence SEQ ID NO: 32 to SEQ ID NO: 36, or any C-terminally cleaved β-amyloid binding product of SEQ ID NO: 36 (namely SEQ ID NO: 37 to SEQ ID NO: 43), or a β-amyloid binding protein comprising SEQ ID NO: 46. A C-terminally cleaved product may be any C-terminal cleavage of SEQ ID NO: 36 wherein the C-terminally cleaved product is capable of binding β-amyloid, is an equivalent stable peptide sequence having peptide stability during expression and production of the fusion protein in mammalian expression system. For example, an ABP variant of the present invention may exhibit improved peptide stability over ABP peptides of the prior art (Ref. WO2006/133566).

The present invention provides an isolated polypeptide that binds β-amyloid, the isolated polypeptide that binds β-amyloid may comprise a sequence selected from the group consisting of consensus sequences SEQ ID NO: 31 or consensus sequence SEQ ID NO: 46 or any β-amyloid binding peptide sequence thereof. For example, the β-amyloid binding peptide of the present invention may comprise a sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, or a sequence comprising an equivalently stable polypeptide sequence.

The present invention provides a fusion protein comprising or consisting of an ABP selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, or a sequence comprising an equivalent polypeptide sequences. In a non-limiting example, the ABP variant may comprise the sequence

(SEQ ID NO: 35; 40 aa) KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI; (SEQ ID NO: 36; 40 aa) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI; or any β-amyloid binding C-terminally cleaved product thereof, such as any peptide comprising or consisting of SEQ ID NO: 46; 29 aa.

A fusion protein of the present invention may comprise an antibody, or fragment thereof, that transmigrates across the blood brain barrier (BBB) (as noted above, BBB is an abbreviation of the antibody carrier that transmigrates the blood brain barrier). The antibody, or fragment thereof, (BBB) may bind surface receptor epitopes on brain endothelial cell that allow for transmigration across the blood brain barrier. For example, such surface receptor epitopes may be TMEM30A or an Insulin-Like Growth Factor 1 Receptor (IGF1R) epitope, or isoforms, variants, portions, or fragments thereof.

The antibody, or fragment thereof, may comprise a sequence selected from the group consisting of:

-   -   an antibody or fragment thereof comprising a complementarity         determining region (CDR) 1 sequence of GFKITHYTMG (SEQ ID NO:1),         a CDR2 sequence of RITWGGDNTFYSNSVKG (SEQ ID NO:2), a CDR3         sequence of GSTSTATPLRVDY (SEQ ID NO:3);     -   an antibody or fragment thereof comprising CDR1 sequence of         EYPSNFYA (SEQ ID NO:4), a CDR2 sequence of VSRDGLTT (SEQ ID         NO:5), a CDR3 sequence of AIVITGVWNKVDVNSRSYHY (SEQ ID NO:6);     -   an antibody or fragment thereof comprising CDR1 sequence of         GGTVSPTA (SEQ ID NO:7), a CDR2 sequence of ITWSRGTT (SEQ ID         NO:8), a CDR3 sequence of AASTFLRILPEESAYTY (SEQ ID NO:9); and     -   an antibody or fragment thereof comprising CDR1 sequence of         GRTIDNYA (SEQ ID NO:10), a CDR2 sequence of IDWGDGGX; where X is         A or T (SEQ ID NO:11), a CDR3 sequence of AMARQSRVNLDVARYDY (SEQ         ID NO:12).

The antibody or fragment thereof may comprise a sequence selected from the group consisting of:

(SEQ ID NO: 13) X₁VQLVX₂SGGGLVQPGGSLRLSCAASGFKITHYTM GWX₃RQAPGKX₄X₅EX₆VSRITWGGDNTFYSNSVKG RFTISRDNSKNTX₇YLQMNSLRAEDTAVYYCAAGST STATPLRVDYWGQGTLVTVSS, where X₁=D or E, X₂=A or E, X₃=F or V, X₄=E or G, X₅=R or L, X₆=F or W, X₇=L or V;

(SEQ ID NO: 18) X₁VX₂LX₃ESGGGLVQX₄GGSLRLSCX₅ASEYPSNFY AMSWX₆RQAPGKX₇X₈EX₉VX₁₀GVSRDGLTTLYADS VKGRFTX₁₁SRDNX₁₂KNTX₁₃X₁₄LQMNSX₁₅X₁₆A EDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTX₁₇V TVSS, where X₁ is E or Q; X₂ is K or Q; X₃ is V or E; X₄ is A or P; X₅ is V or A; X₆ is F or V; X₇ is E or G; X₈ is R or L; X₉ is F or W; X₁₀ is A or S; X₁₁ is M or I; X₁₂ is A or S; X₁₃ is V or L; X₁₄ is D or Y; X₁₅ is V or L; X₁₆ is K or R; and X₁₇ is Q or L;

(SEQ ID NO: 21) X₁VX₂LX₃ESGGGLVQX₄GGSLRLSCX₅X₆S GGTVSPTAMGWX₇RQAPGKX₈X₉EX₁₀VX₁₁ HITWSRGTTRX₁₂ASSVKX₁₃RFTISRDX₁₄ X₁₅KNTX₁₆YLQMNSLX₁₇X₁₈EDTAVYYCA ASTFLRILPEESAYTYWGQGTX₁₉VTVSS, where X₁ is E or Q; X₂ is K or Q; X₃ is V or E; X₄ is A or P; X₅ is A or E; X₆ is V or A; X₇ is V or F; X₈ is G or E; X₉ is L or R; X₁₀ is F or W; X₁₁ is G or S; X₁₂ is V or Y; X₁₃ is D or G; X₁₄ is N or S; X₁₅ is A or S; X₁₆ is L or V; X₁₇ is K or R; X₁₈ is A or S; and X₁₉ is L or Q; and

(SEQ ID NO: 24) X₁VX₂LX₃ESGGGLVQX₄GGSLRLSCAASGRT IDNYAMAWX₅RQAPGKX₆X₇EX₈VX₉TIDWGD GGX₁₀RYANSVKGRFTISRDNX₁₁KX₁₂TX₁₃ YLQMNX₁₄LX₁₅X₁₆EDTAVYX₁₇CAMARQSR VNLDVARYDYWGQGTX₁₈VTVSS, where X₁ is E or Q; X₂ is K or Q; X₃ is V or E; X₄ is A or P; X₅ is V or S; X₆ is D or G; X₇ is L or R; X₈ is F or W; X₉ is A or S; X₁₀ is A or T; X₁₁ is A or S; X₁₂ is G or N; X₁₃ is M or L; X₁₄ is N or R; X₁₅ is E or R; X₁₆ is P or A; X₁₇ is S or Y; and X₁₈ is Q or L.

In specific non-limiting embodiments, the antibody, or fragment thereof, may comprise a sequence selected from any one of:

(SEQ ID NO: 14) DVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMG WFRQAPGKEREFVSRITWGGDNTFYSNSVKGRFTI SRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTAT PLRVDYWGKGTQVTVSS; (SEQ ID NO: 15) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMG WVRQAPGKGLEWVSRITWGGDNTFYSNSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAAGSTSTAT PLRVDYWGQGTLVTVSS (SEQ ID NO: 16) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMG WVRQAPGKGLEWVSRITWGGDNTFYSNSVKGRFTI SRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTAT PLRVDYWGQGTLVTVSS; (SEQ ID NO: 17) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMG WFRQAPGKGLEFVSRITWGGDNTFYSNSVKGRFTI SRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTAT PLRVDYWGQGTLVTVSS; (SEQ ID NO: 19) QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMS WFRQAPGKEREFVAGVSRDGLTTLYADSVKGRFTM SRDNAKNTVDLQMNSVKAEDTAVYYCAIVITGVWN KVDVNSRSYHYWGQGTQVTVSS; (SEQ ID NO: 20) EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMS WFRQAPGKEREFVSGVSRDGLTTLYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWN KVDVNSRSYHYWGQGTLVTVSS; (SEQ ID NO: 22) QVKLEESGGGLVQAGGSLRLSCEVSGGTVSPTAMG WFRQAPGKEREFVGHITWSRGTTRVASSVKDRFTI SRDSAKNTVYLQMNSLKSEDTAVYYCAASTFLRIL PEESAYTYWGQGTQVTVSS; (SEQ ID NO: 23) QVQLVESGGGLVQPGGSLRLSCAVSGGTVSPTAMG WFRQAPGKGLEFVGHITWSRGTTRYASSVKGRFTI SRDNSKNTVYLQMNSLRAEDTAVYYCAASTFLRIL PEESAYTYWGQGTLVTVSS; (SEQ ID NO: 25) QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMA WSRQAPGKDREFVATIDWGDGGARYANSVKGRFTI SRDNAKGTMYLQMNNLEPEDTAVYSCAMARQSRVN LDVARYDYWGQGTQVTVSS; (SEQ ID NO: 26) QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMA WVRQAPGKGLEWVATIDWGDGGTRYANSVKGRFTI SRDNSKNTMYLQMNSLRAEDTAVYYCAMARQSRVN LDVARYDYWGQGTLVTVSS; and

-   -   a sequence substantially identical to any of the above         sequences.

The BBB may be an antibody, or fragment thereof, in an embodiment, the BBB may be a single-domain antibody (sdAb). The sdAb may be humanized.

In an embodiment, the antibody or fragment thereof (BBB) may be linked to an Fc, or fragment thereof, wherein the BBB-Fc construct may form a dimer. The invention further provides a fusion peptide comprising BBB-Fc-L-ABP, wherein the BBB-Fc portion of the fusion peptide is linked to the ABP via a short peptidic linker (for example a linker having less than 12 amino acids) and the Fc or Fc fragment allows for the dimerization of said fusion peptides to provide dimers, which accordingly protects the construct from degradation and increases its serum half-life. The Fc fragment may be any suitable Fc fragment, selected in order to impart desirable pharmacokinetics, in which the Fc or Fc fragment contributes to the long half-life of the fusion molecule. Other Fc or Fc fragment embodiments may modulate, modify or suppress an immunological effector function (Shields et al., 2001). Other Fc fragment embodiments may mediate clearance of the fusion peptide from the brain (Caram-Salas N, 2011). In a non-limiting example Fc or Fc fragments may be Fc mouse Fc2a, or a human Fc1, selected from any one of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and a sequence substantially identical thereto with attenuated effector function, which, when included in said fusion peptide, may have enhanced clearance of amyloid from the brain.

In a compound of the present invention, the BBB may be linked to the ABP via an Fc fragment, and/or any additional suitable linker L.

A compound of the present invention comprises a fusion protein; wherein the fusion protein comprises an antibody or fragment thereof, an Fc fragment, and peptide that binds β-amyloid. The fusion protein may comprise an antibody or fragment thereof linked to the N-terminus of Fc fragment, and the peptide that binds β-amyloid is linked to the C-terminus of the Fc fragment via L, a linking peptide or a chemical linker. The antibody or fragment thereof may be linked to the C-terminus of Fc fragment and the peptide that binds β-amyloid linked to the N-terminus of the Fc fragment.

Accordingly, the present invention provides fusion proteins comprising a single-chain polypeptide comprising an antibody or fragment thereof selected from the group consisting of SEQ ID NO:13 to SEQ ID NO: 26; a peptide that binds ß-amyloid selected from the group consisting of SEQ ID NO: 27 to SEQ ID NO:47; linked via an Fc fragment selected from the group consisting of SEQ ID No: 48 to SEQ ID NO:50. The provided Fc may comprise an Fc with attenuated effector functions. The single-chain polypeptide may form dimers, wherein said dimers may be homodimers or heterodimers with respect to ABP, wherein said homodimers comprise functional ABP and wherein said heterodimers comprise at least one functional ABP.

For example, the fusion protein may comprise SEQ ID NOs: 51 to SEQ ID NO: 70, or a sequence substantially identical thereto. The fusion protein may be a single chain polypeptide, and the single chain polypeptide of the fusion protein may for a dimeric polypeptide. It is noted that the (GGGSGGGGS or GGGGSGGGGS) linker provided in the fusion protein comprised in SEQ ID NO: 51 to SEQ ID NO: 70 may be any suitable linker sequence. For example, the linker sequence highlighted (ex. GGGGSGGGGS) may be any equivalent peptide linking sequence that allows for the linking of the components of the fusion protein provided, as exemplified in SEQ ID NO: 71, wherein the linker may be any peptide or chemical linker.

In an embodiment, the BBB may be linked to an Fc fragment, thus forming a dimer. The Fc fragment may be any suitable Fc fragment, for example mouse Fc2a or human Fc1, with attenuated effector function (Shields et al., 2001).

An ABP of the present invention may comprise sequence SEQ ID NO: 31, for example, the ABP peptide may be a sequence selected from any one of SEQ ID NO: 32 to 36, or a C-terminally cleaved functional product of SEQ ID NO: 36, specifically, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or a peptide of SEQ ID NO: 46 wherein any one of the provided ABP peptides is a functional β-amyloid binding peptide. An ABP as provided herein exhibits unexpected and significant advantages over ABP polypeptides of the prior art. More specifically, the present ABP provided exhibit increased stability and bio-manufacturability. Moreover, a compound or composition of the present invention comprising an ABP, as provided herein, when coupled to BBB, via a linker and/or Fc fragment as provided herein, exhibits a synergistic and unexpected efficacy in transmigrating the blood brain barrier. Such an ABP variant, when coupled to BBB, via a selected Fc fragment as provided herein, provides an unexpected, rapid and improved clearance of Aβ from the brain. Moreover, a fusion protein of the present invention comprising an ABP variant as provided herein exhibits a synergistic and unexpected efficacy in transmigrating the BBB and improved clearance of Aβ from the brain. A fusion protein of the present invention may comprise an ABP of any one of SEQ ID NO: 31 to SEQ ID NO: 47. A composition of the present invention may comprise a mixture of any fusion protein comprising a single-chain peptide or dimer thereof having any ABP of SEQ ID NO: 31 to SEQ ID NO: 47

Accordingly, there is provided a therapeutic composition comprising a blood brain barrier transmigrating antibody or fragment thereof (BBB), an Fc (Fc), linked to a peptide that binds β-amyloid (ABP or ABP variant, or a C-terminal cleaved product thereof), wherein said peptide confers a synergistic increase in stability and efficacy of the therapeutic composition provided. As shown, unexpectedly and most significantly, a single bolus of BBB-Fc-ABP reduced brain Aβ burden by 50% within 24 hrs of treatment (FIG. 9B) compared to three months of multiple treatments with free ABP to achieve similar results in animals, (mouse model of AD) (FIG. 9A). Thus, the compound as provided herein was shown to be far more potent than free ABP in reducing brain Aβ burden. Additionally, this fusion protein comprising Fc provided a substantially longer serum half-life compared to free ABP or BBB-ABP (WO 2006/133566), thereby providing an improved therapeutic compound. (FIGS. 10A-10B).

The ABP variants of the present invention, and fusion proteins (constructs thereof) comprising ABP overcome the disadvantages of the prior art. In the prior art, the linking of ABP with a BBB carrier (WO 2006/133566) alone does not assure the generation of an effective molecule. Fusions comprising an Fc aimed at enhancing serum half-life do not ensure efficient transport of ABP across the blood brain barrier. Specific engineering and formulation of a fusion molecule comprising a BBB carrier-, Fc fragment- and ABP provide an efficient BBB-permeable therapeutic compound. The efficient blood brain barrier-permeable therapeutic compound provided comprises a specifically engineered formulation of a BBB-Fc-ABP, wherein the formulation exhibits a synergistic improvement in compound stability and efficacy. An unexpected increase in the stability of the fusion compositions, and a synergistic increase in efficacy of transmigrating the blood-brain barrier and faster clearance (within 24 hrs) of ß from the brain, as shown in FIGS. 9A-9C, is provided in a composition comprising said fusion protein constructs.

The ABP C-terminal cleavage products provided (eg. SEQ ID NO: 37 to 43 and SEQ ID NO: 46 to 47) herein advantageously provide a specific novel and unexpected subset of functional proteins that allow for an increased yield in an isolatable mixture of functional fusion proteins capable of 3-binding amyloid protein. It is quite advantageous that the fusion proteins of the single-chain polypeptides provided may comprise any one of SEQ ID NO: 31 to 47. It is additionally advantageous that dimers of said single-chain polypeptides may be homodimers, heterodimers, monofunctional heterodimers or any combination thereof. This is particularly important and advantageous in the production of said fusion proteins for use in pharmaceutical compositions, wherein said pharmaceutical composition may comprise any fusion protein provided herein (a homo or heterodimer comprising any one of SEQ ID NO: 53 to SEQ ID NO: 71; wherein the ABP thereof may be any ABP selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 47), or any combination thereof.

The compound provided herein may be referred to as a compound, a fusion protein, formulation, composition or construct. The provided construct may comprise an antibody or fragment thereof (which may be abbreviated BBB), an Fc fragment (abbreviated Fc), and a polypeptide that binds β-amyloid (abbreviated ABP). The construct or composition provided (which may be abbreviated herein as BBB-Fc-ABP or BBB-Fc-L-ABP), comprises components that synergistically overcome the deficiencies encountered in the prior art with respect to blood brain barrier transmigration, efficacy and compound stability. Accordingly, compounds provided herein comprise a novel formulation having superior and unexpected efficacy in transmigrating the blood brain barrier, therapeutic efficacy and compound stability.

There are instances in the prior art where Fc fusions may increase serum half-life, although serum half-life is not necessarily increased in Fc fusions. Moreover, Fc fusions do not necessarily improve transmigration across the blood brain barrier.

The present invention provides a peptide that binds β-amyloid, the peptide sequence may be selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47 or a sequence substantially identical in peptide stability or a β-amyloid binding C-terminally cleaved functional product thereof.

The present invention provides a peptide that binds β-amyloid, wherein when the peptide sequence is referred to as an ABP or an ABP variant, or C-terminally cleaved β-amyloid binding functional product thereof, wherein the ABP may comprise SEQ ID NO: 31 or SEQ ID NO: 46, and may be selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47 and sequence substantially identical in peptide stability or β-amyloid binding activity, for example, any sequence variant of SEQ ID NO:31 or SEQ ID NO:46. An ABP variant having a consensus sequence of SEQ ID NO: 31 or SEQ ID NO: 46 overcomes the disadvantages of the prior art with respect to polypeptide stability. Moreover, a compound comprising an ABP of the present invention exhibits improved compound stability and bio-manufacturability. Furthermore, a compound comprising an ABP or ABP variant as provided herein exhibits a synergistic and unexpected therapeutic improvement.

An ABP of the present invention may comprise a sequence selected from any one of SEQ ID NO: 31 to SEQ ID NO: 47, and an equivalently stable polypeptide sequence or a C-terminal cleavage product provided that β-amyloid binding activity is maintained. An ABP variant of the present invention is superior over ABP sequences of the prior art, wherein the present ABP variant polypeptides exhibit improved stability (as shown in comparison between FIG. 2A and FIG. 16 ).

The present invention also provides fusion proteins (also referred to as compounds, constructs, or fusion molecules) comprising an ABP, or ABP variant, of the present invention. The fusion protein may comprise an Fc fragment. The fusion protein may comprise an antibody or fragment thereof that transmigrates the blood brain barrier, an Fc fragment, and a β-amyloid binding polypeptide sequence that may be selected from the group consisting of SEQ ID NO: 31, to SEQ ID NO: 36, or a C-terminally cleaved product of SEQ ID NO: 36, namely SEQ ID NO: 37-43, or an ABP comprising of or consisting of SEQ ID NO: 46 (eg. SEQ ID NO: 47). The provided BBB-Fc-ABP formulations exhibit synergistic improvement in compound stability and therapeutic efficacy in removing toxic amyloid from the brain.

The fusion protein or construct, which may be referred to herein as BBB-Fc-ABP, may vary in orientations with respect to the components comprised therein. In a compound of the present invention the fusion protein forms a dimer (as shown in FIGS. 1Ai-1D) wherein the fusion peptide dimerizes via the Fc region. For example, in an embodiment, the provided compound may comprise a fusion protein comprising a BBB linked to the N-terminus of the Fc fragment (Fc) and an ABP or ABP variant (ABP) linked to the C-terminus of the Fc fragment via a short peptidic linker attached to the C-terminus (FIGS. 1Ai-1Aiii, wherein the compound is shown as a dimer of the fusion protein), as further shown in FIGS. 1Ai, 1Aii, and 1Aiii where the dimer, as represented in FIG. 1Ai, is a bifunctional homodimer with two functional ABP peptides (e.g., any β-amyloid binding ABP peptide, such as SEQ ID NO: 36), or a bifunctional heterodimer as shown in FIG. 1Aii where the dimer has two different functional β-amyloid binding ABP peptides (ABP and/or C-terminally cleaved ABP, e.g., SEQ ID NO: 37 to SEQ ID NO: 43), or a monofunctional heterodimer with only one functional β-amyloid binding ABP peptide (ABP or C-terminally cleaved ABP, FIG. 1Aiii), the other arm having a non-functional peptide. It is noted that in configuration FIGS. 1Ai to 1Aiii, the N-terminus (fusion) or C-terminus (chemical linking) of ABP or ABP variant may be fused/linked to Fc. In a further embodiment, the provided compound may comprise a BBB linked to the C-terminus of the Fc fragment (Fc) wherein the ABP or ABP variant (ABP) may be linked to the N-terminus of the Fc fragment via a suitable linker (L) (FIG. 1B). In other possible configurations, the BBB may be linked to the N-terminus of the ABP, and the ABP linked to the N-terminus of the Fc fragment (FIG. 1C). In yet another possible configuration the BBB may be linked to the C-terminus of the ABP, and the ABP is linked to the C-terminus of the Fc fragment (FIG. 1D).

The compounds provided herein comprise fusion proteins comprising a single-chain polypeptide sequence selected from any one of SEQ ID NO:51 SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO: 54, SEQ ID NO:55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and a sequence substantially identical to any of these sequences, or dimers thereof. The fusion protein of the present is a fusion protein (BBB-Fc-ABP or BBB-Fc-L-ABP, wherein L may be any suitable linker) dimer (as shown in FIGS. 1Ai-1D, and preferably, FIG. 1Ai-1Aiii or 1B). In a non-limiting example, the compound or fusion protein provided may comprise or consist of a polypeptide comprising sequence SEQ ID NO:35 (ABP-GG-G) or SEQ ID NO:36 (ABP-6G) or a C-terminally cleaved functional product thereof (SEQ ID NO: 37 to 43); an antibody or fragment thereof comprising sequence SEQ ID NO:17 (FC5-H3); and an Fc fragment comprising sequence SEQ ID NO:49 (hFc1X7).

In a specific non-limiting embodiment of the present invention, the compound may comprise sequence SEQ ID NO:55 [FC5-H3-hFc1X7-L-ABP(GG-G)] or SEQ ID NO:56 [FC5-H3-hFc1X7-L-ABP(6G)] or any C-terminally cleaved ABP functional product thereof, wherein the L may be any suitable linker.

The compounds of the present invention transmigrate the blood-brain barrier.

The present invention encompasses a nucleic acid molecule encoding any compound of the present invention as described herein. Vectors comprising the nucleic acid molecule of a fusion protein or compound of the present invention are also included in the scope of the present invention.

The present invention encompasses a composition comprising a compound or fusion protein of the present invention and a pharmaceutically-acceptable carrier, diluent, or excipient.

Kits comprising a pharmaceutical composition of the present invention are also included in the scope of the present invention.

The composition of the present invention may be used for treating Alzheimer's disease in a patient.

The composition of the present invention may be used for reducing toxic ß-amyloid (Aß) levels in the brain or CSF of a subject having increased levels of brain Aß.

The present invention provides a method of treating Alzheimer's disease, wherein a pharmaceutical composition of the present invention may be administered to a subject in need thereof.

The present invention provides a method of reducing toxic ß-amyloid (Aß) levels in the brain of a subject having increased levels of brain Aß. The method of the present invention comprises the administration of a compound of the present invention to a patient with AD. More specifically, the present method comprises the steps of repeated parenteral administration of a sufficient amount of a pharmaceutical composition of the present invention to a subject.

In the method of the present invention, parenteral administration is subcutaneous or intravenous administration.

The method of the present invention reduces toxic ß-amyloid levels, after repeated parenteral administration of a composition provided herein, in the brains of subjects having increased brain levels of Aß. More specifically, toxic ß-amyloid levels are reduced within four weeks of repeated parenteral administration of the composition of the present invention.

The method of the present invention reduces toxic ß-amyloid levels in the cerebrospinal fluid (CSF) of subjects after parenteral administration of the composition of the present invention. More specifically, toxic ß-amyloid levels in the cerebrospinal fluid (CSF) of subjects is significantly reduced within 24 hours of a single parenteral administration of the composition of the present invention; wherein significant reduction is up to 50% within 24 hours.

The present invention provides a blood brain barrier-permeable single domain antibody (sdAb), wherein the sdAb is either camelid FC5 or a humanized version thereof. FC5 displayed in bivalent format on Fc has been shown to have improved blood brain barrier-crossing properties compared to FC5 in V_(H)H format (Farrington et al., 2014).

The present invention provides a compound comprising a BBB that facilitates blood brain barrier transmigration in vitro and an Fc fragment and increases serum half-life of the fusion molecule, wherein serum half-life of the Fc fusion becomes similar to that of a full IgG. Prolonged serum half-life of FC5-Fc-ABP also increases overall brain exposure, which is particularly important for treating chronic diseases such as Alzheimer's Disease.

The FC5-Fc-ABP fusion molecule as well as a humanized version FC5(H3)-hFc-ABP, and IGF1 R5-H2-ABP fusion molecules are single-chain polypeptides produced in CHO cells as dimers. The ABP variants of the present invention, as provided in SEQ ID NO: 31 and SEQ ID NO: 46, are methodically re-engineered with specific point-mutations or deletions that enhance bio-manufacturability and stability of the fusion molecule (SEQ ID NO. 54, SEQ ID NO. 55; SEQ ID NO. 56, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO. 69 SEQ ID NO: 70, SEQ ID NO: 71). The ABP provided may be a C-terminally cleaved species of SEQ ID NO: 36 or may comprise any sequence of SEQ ID NO: 46 The dimers of said single-chain polypeptides may be homo or heterodimers, provided there is at least one functional ABP comprising a sequence of SEQ ID NO: 31, a C-terminal cleavage product thereof or a sequence of SEQ ID NO: 46. The fusion molecules of the present invention comprising the ABP retain amyloid-β (Aβ) binding ability, transmigrate the blood brain barrier, and advantageously allow for the clearance of Aβ from the brain, for example, the rapid clearance of Aβ from the brain within 24 hours of administration.

The ABP variants of the present invention, for example ABP(GG-G) (SEQ ID NO: 35), or ABP(6G) (SEQ ID NO: 36) or any C-terminal cleavage β-amyloid binding product thereof (i.e. an ABP comprising or consisting of sequence of SEQ ID NO: 46), were specifically engineered to enhance stability and manufacturability of the construct (as shown in FIG. 16 ). The enhanced stability and bio-manufacturability of the construct of the present invention is a significant advantage in the present art. The present invention accordingly encompasses ABP and ABP variants that enhance stability and bio-manufacturability of the fusion molecules provided, but also retain therapeutic activity (as provided in FIG. 15A). The C-terminal cleavage products provided herein, wherein said cleavage products comprise a sequence of SEQ ID NO: 46, allow for an mixture of functional fusion proteins comprising a mixture of functional ABP peptides, wherein said mixture advantageously increases the yield of functional product.

Therefore, the fusion molecules comprising different ABP variants, as provided in SEQ ID NO:31, such as in SEQ ID NOs: 32-47 retain the Aβ oligomer binding ability of the parent ABP in vitro (FIGS. 2B, 2C, 12A-12B and 15A-C, 23A-B) as well as the BBB-penetrating property of the parent FC5 both in vitro and in vivo (FIGS. 4A-4D, 5, 6A-6B, 7, 17A-17B, 18 1.A-1.C and 18 2.A-2.C, 19 1.A-1.B and 19 2.A-2.B, 22A-B, 24A-24B, and 25). The ABP is transported across the BBB by the carrier, for example FC5, in vitro and in vivo, as established by its presence in the rat and dog CSF (FIGS. 5 and 6A-6B). The ABP fusion was also transported and delivered to the target regions in the brain, such as hippocampus and cortex in both wild type and AD transgenic mice (FIGS. 8A-8B, 19 1.A-1.B and 19 2.A-2.B, and 24A-24B). The brain-delivered ABP also promoted Aβ clearance in the CSF (FIGS. 9C and 11A and 11B) and in cortical and hippocampal regions of AD transgenic mice and rat (FIGS. 9B and 11C). In addition, and most importantly, Aß clearance in rat AD model correlated with enhanced hippocampal volume and neuronal connectivity (FIGS. 12A and 12B) indicating desired pharmacological/physiological response to treatment.

Unexpectedly and most significantly, a single bolus of BBB-Fc-ABP reduced brain Aβ burden by 50% within 24 hrs of treatment (FIG. 9B) compared to three months of multiple treatments with free ABP to achieve similar results in animals (FIG. 9A). Thus, the BBB-enabled ABP was shown to be far more potent than free ABP in reducing brain Aβ burden. Additionally, this compound has a substantially longer serum half-life compared to free- or FC5-ABP, an essential characteristic of a better therapeutic, due to its fusion with Fc (FIGS. 10A-10B).

In the prior art, the linking of ABP with a BBB carrier (WO 2006/133566) alone does not ensure the generation of an effective molecule. Fusion with Fc to enhance serum half-life also does not ensure efficient transport of ABP across blood brain barrier. Suitable engineering and formulation of BBB carrier-, Fc fragment- and ABP fusion molecule, as provided in the fusion protein constructs of present invention, provide an efficient BBB-permeable therapeutic compound.

This novel bi-functional fusion molecule has distinct advantage over conventional therapeutic antibodies currently under development. First, the BBB-fused therapeutic ABP can penetrate the brain at much higher levels and at a faster rate, which substantially improves the therapeutic efficacy. In addition, ABP and BBB (ex. FC5) have relatively lower affinity towards their respective receptors and thus are likely cleared from the brain faster, and accordingly facilitate faster clearance of ABP-bound Aβ. Unlike therapeutic antibodies that mainly employ reactive microglia/astrocytes for Aβ clearance (e.g., aducanumab), which is a slower process, the fusion molecule of the present invention likely employs faster perivascular drainage pathway for Aβ clearance. This is supported by a nearly 50% reduction in CNS Aβ within 24 hr of treatment compared to free ABP (FIG. 9B) and antibody-based therapeutics, which require months of treatment with repeated multiple doses to achieve similar results.

Moreover, the present compound is less likely to elicit neuro-inflammatory response compared to antibody-based therapeutics.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will now be described by way of example, with reference to the appended drawings, wherein:

FIGS. 1Ai-1D: show schematic drawings of a blood brain barrier-crossing, amyloid-binding fusion protein dimer. The schematic of a single-chain polypeptide fusion protein comprising a BBB-crossing single-domain antibody (BBB or BBB carrier), an Fc fragment (Fc) and an amyloid-binding peptide (ABP) fused via a linker peptide is provided and shown as a dimer. FIGS. 1Ai-1D show the corresponding dimers of the fusion protein comprising BBB-Fc-ABP. The dimer of the fusion protein could be a bifunctional homodimer or bifunctional heterodimer with respect to ABP in that both ABP arms contain functional ABP peptides (e.g., SEQ ID No: 36, FIG. 1Ai) and/or various C-terminally cleaved functional ABPs (e.g., SEQ ID No: 36 and/or SEQ ID No: 37 to SEQ ID No: 43, FIG. 1Aii,) or a monofunctional heterodimer wherein one of the ABP arms is functional (ABP or a C-terminally cleaved functional cleavage product thereof) and the other arm is non-functional peptide (FIG. 1Aiii). The components of the single-chain polypeptide (i.e. BBB, Fc, and ABP) are depicted in various configurations, as illustrated in FIGS. 1Ai-1Aiii, 1B, 1C, and 1D.

FIGS. 2A-2C: show the production of FC5-mFc-ABP and humanized FC5 (H3)-hFc-ABP (ABP SEQ ID NO: 32) fusion molecule in CHO cells. As shown in FIG. 2A, a Coomassie blue stained gel after separation of FC5 fusion molecules by SDS-PAGE (NR—non-reducing and R—reducing conditions) showing successful production of recombinant fusion molecule. FIG. 2B and FIG. 2C: Aß-oligomer binding of free ABP and BBB-Fc-ABP fusion protein by ELISA and Western blot (WB) Overlay assay. Free or fused ABP was immobilized on ELISA plate and exposed to Aß. Bound Aß was detected with Aß-specific antibody 6E10 or 4G8. The fusion molecules were also separated by SDS-PAGE, transferred to PVDF paper and exposed to Aß. Bound Aß was detected with specific antibody as above. Results show that ABP retained its Aß oligomer binding ability after fusion with the BBB carrier. Mo: Aß monomers; Oli: Aß Oligomers

FIG. 3 : shows an immunohistofluorescence assay of the binding of FC5-Fc-ABP to amyloid deposits in AD-Tg mice (B6.Cg-Tg, Jackson Lab). ABP retains the ability to bind naturally produced Aß aggregates in AD-Tg mouse brain as shown by immunohistofluorescence assay. Brain sections from wild type and AD-transgenic mice were incubated with IR 800-labelled FC5-mFc-ABP and the bound fusion molecule was visualized under fluorescence microscope. Selective binding (bright spots) were seen in brain sections from AD-Tg mice that produce Aß deposits and not in brain sections from wild type mice that does not produce amyloid deposits.

FIGS. 4A-4D: show BBB permeability of FC5 is retained after fusion with ABP in vitro. Blood brain barrier crossing of FC5-ABP fusion molecules was assessed in in vitro BBB models from rat and human. Fusion molecules crossing BBB were detected by nanoLC-MRM method (FIG. 4A, FIG. 4B and FIG. 4C) and by Western blot analysis using Fc-specific antibody (FIG. 4D, done in triplicate). FC5-mFc-ABP crossed the BBB as effectively as FC5-mFc, whereas Fc-ABP without the BBB carrier moiety FC5 did not traverse across the brain endothelial cell monolayer. As expected, control single domain antibodies EG2 and A20.1, or control full IgG (anti-HEL) did not cross the blood brain barrier. Similar results were obtained with humanized FC5-H3-hFc-ABP fusion protein (FIG. 4C and FIG. 4D).

FIG. 5 : shows serum and CSF pharmacokinetics of FC5-Fc-ABP in vivo. FC5-mFc-ABP was administered intravenously into rats via tail vein injection at the indicated doses (2.5, 6.25, 12.5, and 25 mg/kg). Serum and CSF were serially collected. FC5-Fc-ABP levels were quantified using nanoLC-MRM method. As shown in FIG. 5 , FC5-mFc-ABP appeared in the CSF in a time- and dose-dependent manner with Cmax between 12 and 24 h, indicating transport of ABP by FC5 into brain and CSF compartments in vivo. Serum PK parameters (FIG. 5 and Table 1) show that alpha- and beta-half-life of FC5-mFc-ABP is similar to that of a full IgG (a benchmark antibody containing rat Fc).

FIGS. 6A-6B: show serum and CSF PK profile of FC5-mFc-ABP in beagle dog. FC5-mFc-ABP was administered by intravenous injection to 10-12-year old beagle dogs and serum and CSF were serially collected and analyzed by nanoLC-MRM (FIG. 6A) and by Western blot using Fc-specific antibody (FIG. 6B). Asterisks indicates blood-contaminated sample (not shown in MRM analyses). As can be seen, FC5-mFc-ABP appeared in the CSF in a time-dependent manner indicating transport of ABP by FC5 across dog blood brain barrier in vivo. The PK parameters and CSF exposure were analyzed by WinNonlin software and are shown in Table 2 below.

FIG. 7 : shows BBB permeability and CSF appearance of FC5 fused with human Fc (hFc) and chemically linked to ABP (FC5-hFc-ABP) in vivo (rat model). FC5-hFc was linked with ABP-cystamide using a heterobifunctional cross-linker sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) according manufacturer's instructions (ThermoFisher Scientific). Chemically conjugated molecule was administered intravenously into rats via tail vein at 6.25 mg/kg and serum and CSF samples were collected at 4 and 24 hrs and analyzed by nanoLC-MRM. FC5-hFc-ABP appears in the CSF in a time-dependent manner in contrast to Fc-ABP without the BBB carrier. It should be noted that in this chemically linked construct, C-terminus of ABP is linked to Fc fragment (random region) and N-terminus is free, unlike in the fusion construct, wherein it is N-terminus of ABP that is fused to C-terminus of Fc and C-terminus of ABP is free. This reversal in the orientation of ABP did not affect its Aß-binding ability and transport across the blood brain barrier.

FIGS. 8A-8B: show levels of FC5-mFc-ABP measured in various brain regions (cortex and hippocampus) after intravenous injection in mice. 15 mg/kg dose of FC5-mFc-ABP was injected intravenously into the tail vein of either wild type (WT) or AD-transgenic (AD-Tg, B6.Cg-Tg, Jackson Lab) mice and brains were collected at 4 and 24 hrs after intra-cardiac saline perfusion. Hippocampal and cortical tissues were dissected and analyzed by nanoLC-MRM (FIG. 8A) and by Western blot using Fc-specific antibodies (FIG. 8B). Specific peptides belonging to all the three components of the fusion molecule (BBB, in this example FC5, Fc and ABP) were detected by MRM in both cortex and hippocampus, indicating that FC5 carrier successfully delivered ABP to the target areas of the brain. Measured levels ranged between 750-1400 ng/g brain tissue at different time points, compared to ˜50 ng/g tissue typically measured for control single-domain antibody A20.1 fused to Fc, or Fc fragment alone. This was further confirmed by Western blot analysis probing for Fc and ABP in tissue extracts (FIG. 8B). No protein signal of the fusion molecule was detected in animals receiving just saline by Western blot. There was a dose-dependent increase in FC5-mFc-ABP levels detected by Western blot in the target regions of the brain.

FIGS. 9A-9C: shows the effect of ABP on Aß levels in transgenic (Tg) mice: Comparison between treatment with ABP alone (FIG. 9A) or ABP fused with BBB carrier FC5 (FIG. 9B and FIG. 9C). Two different AD Tg mouse models, triple transgenic (3×Tg-AD, sv129/C57BL6 mice harboring PS1M146V, APP_(Swe) and tauP301L transgenes, Dr. F. M. LaFerla, University of California) and double transgenic (B6.Cg-Tg, harboring PSEN1dE9 and APP_(Swe) transgenes, Jackson Lab) were used; mice were dosed subcutaneously (sc) with 300 nmol/kg of free ABP every second day over a 3-month or a 2-month period, respectively. At the end of the treatment period, Aß levels in the brain were measured by ELISA. The treatment with ABP alone resulted in 25-50% reduction in brain Aß after 2-3 months of multiple treatments (every second day) (FIG. 9A). The FC5-mFc-ABP construct was injected intravenously into double-transgenic AD mice (B6.Cg-Tg, 15 mg/kg; equivalent of 220 nmol/kg) and brain Aß levels were measured by both ELISA and nanoLC-MRM 24 h after injection. Unexpectedly, about 50% amyloid reduction was observed within 24 hr of treatment with FC5-mFc-ABP (FIG. 9B), indicating that efficient brain delivery of ABP by FC5, suitably linked to Fc, dramatically increased the efficacy of ABP in reducing brain Aß levels. CSF analysis also indicated a significant decrease in Aß₁₋₄₂ levels within 24 hrs following FC5-mFc-ABP treatment (FIG. 9C). The signature peptide or epitope of Aß detected by MRM or ELISA analyses is remote/different from the Aβ epitope recognized by ABP (therefore, not interfering with its quantification by either ELISA or MRM).

FIGS. 10A-10B: show examples of enhanced serum half-life of FC5-Fc-ABP construct compared to FC5-ABP (i.e. without Fc component): FC5-ABP and FC5-Fc-ABP were injected into rats via tail vein and serial serum samples were collected at various time points and analyzed by direct ELISA with FC5-specific antibody. As can be seen in FIG. 10A, FC5-ABP construct was rapidly cleared in the serum (less than 1 hr) compared to FC5-Fc-ABP (FIG. 10B), indicating substantial increase in the serum stability of the molecule comprising the Fc fragment.

FIGS. 11A-11C: show the effect of FC5-mFc-ABP on brain amyloid burden in Tg rats: AD-Tg rats were dosed with either saline or FC5-mFc-ABP via tail vein every week over a period of four weeks (loading dose of 30 mg/kg and subsequent four weekly doses of 15 mg/kg). CSF levels of FC5-mFc-ABP and Aß were analyzed by nanoLC MRM (FIG. 11A and FIG. 11B). Before and after four weeks of treatment, brain Aß levels were determined by PET scan using a specific Aß-binding agent [18F] NAV4694. Following tracer injection, 60 min Dynamic images were acquired, transmission scans were obtained, images were reconstructed and Binding Potential (BP_(ND)) parametric maps were generated. FC5-mFc-ABP reduced CSF Aß level in rats within 24 hrs (FIG. 11A and FIG. 11B). An inverse relationship between the CSF levels of FC5-mFc-ABP and Aß was observed, as in Tg rats, suggesting target engagement and rapid clearance of Aß by ABP delivered to the brain and CSF by FC5 (FIG. 11B). This was further corroborated by PET scan which clearly indicated a significant reduction (30-50%) of rat brain Aß levels following four weeks of treatment with FC5-mFc-ABP (FIG. 11C).

FIGS. 12A-12B: shows volumetric Magnetic Resonance Imaging (MRI, using Fast Imaging with Steady-state Precession) and functional MRI (fMRI) of Tg rats before and after treatment with saline or FC5-mFc-ABP. As shown in FIG. 12A, increased hippocampal volume was observed in ABP-treated Tg rats (Tg-ABP) compared to saline-treated Tg rats (Tg-Sal) after four weeks of treatment. As shown in FIG. 12B, group comparison after four weeks of treatment showed that the ABP-treated Tg rats (Tg-ABP) had greater Anterior Cingulate Cortex (ACC) connectivity compared to saline-treated Tg rats (Tg-Sal).

FIGS. 13A-13B: show time- and dose-dependent appearance of FC5-mFc-ABP in the CSF of beagle dog and a decrease in CSF Aß levels as seen Tg mice (FIG. 9C) and Tg rats (FIGS. 11A and 11B). FC5-mFc-ABP was administered by intravenous injection to 10-12-year old beagle dogs at 15 mg/kg and 30 mg/kg and serum and CSF were serially collected and analyzed by nanoLC-MRM for FC5-mFc-ABP and Aß levels. As can be seen, FC5-mFc-ABP appeared in the CSF in a time- and dose-dependent manner. Importantly, as seen in Tg mice and Tg rats, there was a significant decrease in CSF Aß levels within 24 hrs after the FC5-mFc2a-ABP injection, suggesting translational nature of FC5 carrier in larger animals and also cross-species efficacy of ABP in reducing CNS Aß burden.

FIG. 14 : shows the generation of ABP fusion molecule with a different BBB carrier. To assess the versatility of ABP fusion molecule, ABP was successfully fused with another humanized BBB carrier IGF1 R5 (H2). As shown, the bi-functionality of the molecule was retained, ABP's ability to bind Aß oligomer (ELISA and overlay assays) and also IGF1 R5's ability deliver ABP across BBB model in vitro (data not shown). This clearly indicates that ABP can be fused to different BBB-crossing single-domain antibodies to be delivered to the brain.

FIGS. 15A-15C: show Aß oligomer binding by different single-domain antibody-Fc-ABP constructs (FIGS. 15A, 15B and 15C). In some of these constructs, ABP has been modified by site-specific mutations or removal of C-terminus portion of the molecules as indicated in the SEQ ID NOs. All constructs retained similar potency in binding Aß oligomers by ELISA method.

FIG. 16 : shows the production of FC5-hFc1X7-ABP with specific mutations to improve stability and bio-manufacturability. FC5-hFc1X7-ABP carrying specific mutation (such as the ABP of SEQ ID NO: 35 or SEQ ID NO: 36) were produced in CHO cells and separated on SDS-PGE under reducing (R) and non-reducing (NR) conditions and stained with Coomassie blue as described in FIG. 2 . Separated protein transferred to nitrocellulose membrane and immunoblotted with either FC5-specific or hFc-specific or ABP-specific antibodies. In another set, Aß-binding of ABP in the fusion molecule was also tested by overlay assay. Bound Aß was detected with Aß-specific antibody 6E10. As can be seen, the methodic modification of ABP with specific mutation substantially enhanced the stability of the molecule generated, as indicated by single protein band under reducing and non-reducing conditions.

FIGS. 17A-17B: show blood brain barrier permeability of various FC5-Fc-ABP constructs and IGF1 R5-Fc-ABP construct in vitro. BBB-crossing was assessed in in vitro rat BBB model as described in FIG. 4 and molecules crossing blood brain barrier were detected by nanoLC-MRM method. All ABP variants fused to humanized FC5 and IGF1R carriers crossed the blood brain barrier effectively. As expected, A20.1, a non-BBB permeable sdAb did not cross blood brain barrier, likewise ABP fused to A20.1 did not cross BBB (FIG. 17A). In FIG. 17B, it is shown that “finger-print” peptides for all the three components of the fusion molecule, FC5, Fc and ABP were detected by nanoLC-MRM, thereby indicating the transmigration of the intact FC5, Fc and ABP across the BBB.

FIGS. 18 1.A-1.C and 18 2.A-2.C: shows humanized FC5(H3)-hFc1X7-ABP construct (ABP, SEQ ID NO: 35 and SEQ ID NO: 36) are transported across in vitro blood-brain barrier intact by FC5. Blood brain barrier-crossing was assessed in in vitro rat BBB model as described for FIG. 4 and molecule crossing BBB was detected by Western blot and ELISA assays. FIGS. 18 1.A and 18 1.B show immunoblots probed with hFc- and ABP-specific antibodies, respectively. The molecular size is exactly identical to that of the fusion molecule that was applied to in vitro blood brain barrier model. FIG. 18 1.C shows sandwich ELISA in which the molecule after crossing the BBB was captured by FC5-specific antibody on ELISA plate and detected with ABP-specific antibody. This sandwich ELISA confirmed that FC5(H3)-hFc1X7-ABP remained intact after crossing rat blood brain barrier in vitro. Similar results were obtained with FC5(H3)-hFc1X7-ABP (ABP, SEQ ID NO: 36); FIGS. 18 2.A-2.C.

FIGS. 19 1.A-1.B and 19 2.A-2.B: show humanized FC5(H3)-hFc1X7-ABP constructs (ABP, SEQ ID NO: 35 and SEQ ID NO: 36) are transported across in vivo blood-brain barrier and delivered to the brain intact by FC5. The FC5-ABP fusion molecule was administered intravenously into wild type and AD-Tg mice via tail vein and brains were collected following intra-cardiac perfusion as described for FIG. 8 . Brain cortex was homogenized and extracted in RIPA buffer and the extract was subjected to Western blot and sandwich ELISA analysis as described for FIGS. 18 1.A-1.C and 18 2.A-2.C. FIGS. 19 1.A and 19 2.A show immunoblot probed with ABP-specific antibody. The molecular size is exactly identical to that of the fusion molecule that was injected into the animals. FIGS. 19 1.B and 19 2.B show sandwich ELISA of the same extract, molecule in the extract captured with FC5-specific antibody on the ELISA plate and detected with ABP-specific antibody. This confirms immunoblot results that FC5(H3)-hFc1X7-ABP is transported across the BBB in vivo and delivered to the brain intact.

FIGS. 20A-20B: show immunohistochemical analysis of ex-vivo binding (FIG. 20A) and in vivo binding (FIG. 20B) of FC5(H3)-hFc1X7-ABP (ABP, SEQ ID NO: 36) to endogenous Aß deposits in AD-Tg mouse brain (B6.Cg-Tg, Jackson Lab). Brain sections from AD-transgenic mice were incubated with FC5(H3)-hFc1X7-ABP (ABP, SEQ ID NO: 36) and the bound fusion molecule was visualized with HRP-conjugated FC5-specific antibody. Selective binding (black spots) was seen with FC5(H3)-hFc1X7-ABP construct but not with FC5(H3)-hFc1X7 without ABP (FIG. 20A), indicating ABP-dependent binding of Aß deposits (target engagement) in the brain. No binding was seen in brain sections from wild type mice that does not produce amyloid deposits (data not shown). Similar binding of Aß deposits were detected (using ABP-specific antibody) following intra-hippocampal injection (4 hrs post-injection) of FC5(H3)-hFc-ABP construct into AD transgenic mice (FIG. 20B).

FIGS. 21A-21B: show target engagement by FC5(H3)-hFc1X7-ABP (ABP, SEQ ID NO: 32) in vivo. FIG. 21A shows binding of Alexa 647-labeled FC5-hFc-ABP construct to natural amyloid-ß(Aß) deposits in AD transgenic mice in vivo after intra-hippocampal injection. Identity of Aß was confirmed by probing the brain sections with Aß-specific antibody 6E10 labelled with Alexa 488 and demonstrating co-localization of two signals (Merge). FIG. 21B shows demonstration of target engagement by ELISA. Following intra-hippocampal injection (4 hrs post-injection) of FC5(H3)-hFc-ABP construct into wild type and AD transgenic mice, hippocampal formation was dissected and homogenized. FC5-ABP fusion construct was detected by sandwich ELISA using FC5 antibody as capturing antibody and ABP antibody as the detection antibody. In vivo binding of ABP to endogenous Aß was detected by the same sandwich ELISA but with Aß-specific antibody as the detection antibody. It is clear from the FIGS. 21A and 21B that the FC5-ABP construct remains intact 4 hrs post-injection in both wild type and AD transgenic mice. Most importantly, in Tg mice which expresses human Aß, injected ABP binds Aß and is pulled down as a complex (FC5(H3)-hFc-ABP*Aß) indicating Aß-target engagement by ABP in vivo.

FIGS. 22A-22B: show PK, PD comparison between non-humanized and humanized FC5-Fc-ABP constructs. FC5-mFc2a-ABP or FC5(H3)-hFc1x7-ABP was administered intravenously into rats via tail vein injection at 15 mg/kg as described for FIG. 5 . Serum and CSF were serially collected. FC5-Fc-ABP levels were quantified using nanoLC-MRM method. As shown in FIG. 22A, serum and CSF PK profile were very similar for non-humanized and humanized constructs. FC5-mFc2a-ABP or FC5(H3)-hFc1x7-ABP was administered intravenously into Tg mice via tail vein injection at 15 mg/kg as described for FIG. 9B. FC5-Fc-ABP and Aß levels in the CSF were measured by nanoLC-MRM as described in FIG. 9B. As shown in FIG. 22B, the levels of non-humanized and humanized FC5-Fc-ABP in the CSF were similar, and most importantly, changes (decrease) in CSF Aβ levels were also very similar, indicating that humanization of FC5-Fc-ABP construct did not affect the PK and PD profile of the fusion construct.

FIGS. 23A-23C: show the Cation-exchange (CEX) chromatography profile of FC5(H3)-hFc1x7-ABP (SEQ ID NO: 56) produced in stable CHO cell line indicating the generation of multiple forms of FC5(H3)-hFc1x7-ABP (ABP variants) during manufacturing. A detailed Western blot analysis with anti-hFc antibody (FIG. 23A) and Mass spectroscopic analysis (FIG. 23B) revealed that different forms of the fusion protein were generated due to the C-terminal cleavage of the ABP peptide at different positions (as illustrated in SEQ ID NO: 37 to SEQ ID NO: 43 of SEQ ID NO: 36) during the production, generating bifunctional dimers, i.e. bifunctional homodimers and heterodimers with respect to ABP functionality (both the ABP arms are functional) and monofunctional heterodimers (one functional ABP arm and another non-functional arm) of the fusion proteins as depicted in FIG. 1Ai-1Aiii. Representative Mass Spectrometric data of CEX fraction of FC5(H3)-hFc1x7-L-ABP (6G) (SEQ ID NO: 56) dimers produced in CHO cells showing the presence of intact and C-terminally cleaved ABP (FIG. 23B). Homodimer of SEQ ID NO: 56 comprising ABP SEQ ID NO: 36 (Intact chain dimer peak), heterodimers comprising C-terminally cleaved ABP variants [SEQ ID NO: 57 (NI, Asn-Ile peak); SEQ ID NO: 60 (RVKNI peak)] and non-functional ABP (ASAQ/ASLA peak) are shown (FIG. 23B). The precise nature of the cleavage products of the fusion protein (SEQ ID NO: 56) was not known and not obvious until detailed analyses were carried out. It is further shown that the ß-amyloid binding activity (Western blot Aß overlay, and ELISA assays, EC 50) of the CEX fraction containing a mixture of homodimers (bifunctional) and heterodimers (mono and bifunctional) (peak2), FIG. 23A, representing about 25% of the total protein) was very similar to that of the bifunctional homo- and hetero dimers (peak3), representing approximately 65% of the total protein). This strongly indicated that the heterodimer and homodimer fractions (middle and the main peak of the CEX) can be combined without significantly losing ß-amyloid binding activity. This advantageously would substantially increase the yield of FC5(H3)-hFc1x7-ABP fusion protein during large-scale biomanufacturing, especially during downstream processing of the fusion protein product. This has been demonstrated in FIG. 23C, which shows that modified CEX conditions generate a fraction with high yield (over 85% of total protein applied on the column) consisting of both homodimers and heterodimers (mono and bifunctional). The ß-amyloid binding activity (EC 50) of this combined pool (fractions B3 to B6) was very similar to other CEX fractions containing either predominantly homodimers (C10-D2, E12) or heterodimer forms (C6-C8).

FIGS. 24A-24B: show the in vivo brain delivery of the FC5(H3)-hFc1x7-ABP fusion protein (mixture of homo- and hetero-dimers) after intra-venous injection (tail vein) of wild-type (wt) and Tg mice (see legend to FIGS. 8A-8B and 19 1.A-1.B and 19 2.A-2.B for details) as assessed by sandwich ELISA (24 A) and western blot analysis (24 B) which is very similar to the brain delivery of predominantly homodimeric form of FC5(H3)-hFc1x7-ABP (SEQ ID NO: 56, see FIGS. 19 1.A-1.B and 19 2.A-2.B), confirming that the heterodimeric form of FC5(H3)-hFc1x7-ABP in the mixture does not affect brain delivery of the fusion protein across the blood-brain barrier in vivo.

FIG. 25 : shows CSF exposure of FC5(H3)-hFc1x7-ABP fusion protein (mixture of homo- and hetero-dimers) after intra-venous injection of Tg mice and its effect on CSF ß-amyloid (see FIGS. 22A-22B and 24A-24B). As shown in FIG. 25 , CSF exposure of FC5(H3)-hFc1x7-ABP and changes (decrease) in CSF Aβ levels are very similar to that seen with predominantly homodimeric form of FC5(H3)-hFc1x7-ABP (SEQ ID NO: 56, see FIGS. 22A-22B), further confirming FC5(H3)-hFc1x7-ABP in the mixture does not affect functional efficacy of FC5(H3)-hFc1x7-ABP fusion protein in vivo.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides polypeptides, fusion proteins comprising said polypeptides, and fusion proteins comprising said polypeptides and antibodies or fragments thereof that transmigrate the blood-brain barrier.

The present invention provides peptides which binds beta-amyloid (ß-amyloid). The peptides (or proteins) that bind β-amyloid may selectively bind pathologically relevant ß-amyloid₁₋₄₂ (Aβ₁₋₄₂) aggregates, and may be abbreviated and referred to herein as ABP or ABP variants (or collectively as ABP), or C-terminally cleaved ABP products.

The present invention provides fusion proteins comprising ABP (ABP variants and C-terminal cleavage products thereof) linked to an antibody or fragment thereof that crosses the blood-brain barrier. In an embodiment, the fusion protein comprising ABP and a BBB additionally comprises an Fc or fragment thereof, wherein the ABP and BBB components of the fusion protein may be linked via an Fc region or portion thereof to form a single-chain polypeptide (abbreviated herein as BBB-Fc-ABP or BBB-Fc-L-ABP). For example, a construct of the present invention may comprise BBB-Fc-ABP or BBB-Fc-L-ABP, wherein L may be any suitable linker. The BBB-Fc-ABP (also abbreviated BBB-Fc-L-ABP) construct provided may be a single chain polypeptide or a dimeric polypeptide thereof, wherein the single-chain polypeptide comprising BBB-Fc-ABP may form a multimer (preferably a dimer) via the component Fc region, the multimer may be a homodimer or heterodimer with respect to ABP in the fusion protein, the heterodimer may be monofunctional or bifunctional with respect to ABP having one functional ABP arm or having two different functional ABP arms respectively.

The present invention relates to compounds that transmigrate the blood-brain barrier, and uses thereof. More specifically, the present invention relates to compounds comprising a BBB and an ABP and their use in the treatment of Alzheimer's disease (AD).

There is a need for therapeutic formulations that can efficiently transmigrate ABP across the blood brain barrier, and provide the clearing of Aβ through the binding of ABP. In the prior art, a 40-amino acid Aβ-binding peptide (ABP) was identified that selectively binds Aβ₁₋₄₂ oligomers implicated in AD development (WO 2006/133566). This Aβ-binding peptide inhibits Aβ binding to cellular proteins and inhibits Aβ₁₋₄₂-induced cell toxicity in vitro (Chakravarthy et al, 2013). This Aβ-binding peptide binds amyloid deposits in AD transgenic mice brain, as well as binds amyloid deposits in the brains from AD patients in vitro. More importantly when directly injected into live AD transgenic mice brain (Chakravarthy et al, 2014) ABP targets natural amyloid deposits in vivo. Thus, ABP can potentially target CNS Aβ, can assist in clearing Aβ from the brain, and reduce its toxic effect. However, systemically-administered ABP has limited ability to cross BBB and access the brain parenchyma by itself.

Accordingly, although ABP has been shown to bind Aβ deposits when directly applied, in order to bind and clear Aβ from the brain, parenterally administered ABP needs to permeate the blood brain barrier. The present invention advantageously provides an ABP fused to a BBB-permeable single-domain antibody, such as FC5 or IGF1R, via an Fc fragment to provide a bi-specific blood brain barrier-permeable therapeutic (Farrington et al, 2014). The BBB-Fc-ABP construct of the present invention may dimerize, i.e. the BBB-Fc-ABP single-chain fusion protein may form dimers of two single chain fusion proteins to yield a dimeric compound wherein each single chain of the dimer comprises a BBB, an Fc fragment and an ABP, to provide a BBB-Fc-ABP dimer. The BBB-Fc-ABP or BBB-Fc-L-ABP construct and dimers thereof, allows for the efficient transmigration of ABP across the blood brain barrier. Accordingly, the advantageous therapeutic clearing of Aβ through the binding of ABP in CSF and brain parenchyma is provided in the constructs and methods of the present invention.

In order to enable brain delivery of ABP and improve its efficacy, the ABP peptide is presently fused to the C-terminus of an Fc fragment, whereas a BBB-permeable single-domain antibody, such as FC5 (WO 2002/057445), is fused to the N-terminus of the same Fc fragment, to create a bi-specific BBB-permeable therapeutic (Farrington et al, 2014). In a non-limiting embodiment of the present invention, the Fc fragment may be mouse (SEQ ID NO. 48) or human (SEQ ID NO: 49; SEQ ID NO: 50). In an embodiment, the Fc fragment of the present invention was engineered to reduce effector functions (Shields et al., 2001). For example the Fc fragment may be hFc1x7 (SEQ ID NO:49) wherein the Fc fragment in the BBB-Fc-ABP fusion protein advantageously allows for the dimerization of the fusion protein to yield a therapeutically effective fusion molecule (BBB-Fc-ABP dimer) capable of transmigrating the blood brain barrier. In an embodiment, the BBB-Fc-ABP fusion protein may be FC5-H3-hFc1x7-ABP(6G) (SEQ ID NO: 56) and dimers thereof, or functionally equivalent fusion proteins of SEQ ID NO:56, namely SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 comprising C-terminally cleaved ABP products capable of binding β-amyloid protein. The fusion protein may comprise a linker sequence L that is as embodied in SEQ ID NO: 56 or SEQ ID NO:71, or any suitable linker sequence.

The present invention provides an isolated peptide which binds beta-amyloid (ß-amyloid). A peptide of the present invention may comprise or consist of a sequence.

(SEQ ID NO: 31; 40 aa) X₁TFX₂TX₃X₄ASAQASLASKDKTP KSKSKKX₅X₆STQLX₇SX₈VX₉NI

-   -   where X₁=K, G or A, X₁=G or A, X₂=K, G or V, X₃=R, G or A, X₄=K,         G or A, X₅=R, G or V, X₆=N, G or V, X₇=K, G or V, X₈=R, G or A,         X₉=K, G or A, or a β-amyloid binding C-terminal cleavage product         thereof.

There present invention provides a C-terminal cleavage product of SEQ ID NO: 36 (40 aa), namely SEQ ID NO: 37 (38 aa) to SEQ ID NO: 43 (32 aa). The present invention provides specific C-terminal deletions and mixtures comprising these C-terminal deletions. Specifically, SEQ ID NO: 36 may be cleaved at 1 amino acid from the C-terminus [−1 aa to yield a 39 amino acid cleavage product by the removal of amino acid Isoleucine (I)]; or it may be cleaved 2aa from the C-terminus (−2aa deletion of NI, ie. the removal of NI (Asparagine and Isoleucine); or it may be cleaved at 3 amino acid from the C-terminus (minus KNI); or it may be cleaved 4 aa from the C-terminus (removal of VKNI); or it may be cleaved at 5 amino acid from the C-terminus (minus RVKNI); or it may be cleaved at 6 amino acid from the C-terminus (minus SRVKNI); or it may be cleaved at 7 amino acid from the C-terminus (minus KSRVKNI); or it may be cleaved at 8 amino acid from the C-terminus (minus LKSRVKNI). The provided cleavage products yield functional (β-amyloid binding) peptides that may be comprised in the fusion proteins provided, wherein the fusion proteins provided may be homo or heterodimers, and the homo and heterodimers comprise at least one functional ABP arm (in the case of a homodimer, both arms are functional), or a mixture comprising any combination thereof. The fusion protein dimer of the present invention may comprise a single-chain polypeptide of any one of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, or any combination thereof, or a dimer having at least one of the defined single-chain polypeptides.

The present invention provides a β-amyloid binding peptide comprising or consisting of SEQ ID NO: 46 (29 aa).

An ABP peptide (ABP, ABP variant or a C-terminal cleaved product thereof) of the present invention may be selected from the group consisting of: SEQ ID NO:27 to SEQ ID NO:36 or a sequence substantially identical thereto, or a C-terminally cleaved ABP peptide capable of binding β-amyloid protein, such as SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43. In an embodiment, the polypeptide provided is an ABP variant comprising a sequence substantially equivalent to SEQ ID NO: 31, or a biologically functional C-terminal cleavage product thereof, or any ABP comprising sequence SEQ ID NO: 46. A sequence that is substantially equivalent thereto may confer equivalent stability to the fusion molecules.

The present invention provides a compound, namely a fusion protein, comprising an antibody or fragment thereof that transmigrates across the blood brain barrier (BBB) and a polypeptide that binds β-amyloid. The present invention provides fusion proteins comprising a BBB, a polypeptide that binds β-amyloid, and an Fc.

The term “antibody”, also referred to in the art as “immunoglobulin” (Ig), as used herein refers to a protein constructed from paired heavy and light polypeptide chains; various Ig isotypes exist, including IgA, IgD, IgE, IgG, and IgM. When an antibody is correctly folded, each chain folds into a number of distinct globular domains joined by more linear polypeptide sequences. For example, the immunoglobulin light chain folds into a variable (V_(L)) and a constant (C_(L)) domain, while the heavy chain folds into a variable (V_(H)) and three constant (C_(H), C_(H2), C_(H3)) domains. Interaction of the heavy and light chain variable domains (V_(H) and V_(L)) results in the formation of an antigen binding region (Fv). Each domain has a well-established structure familiar to those of skill in the art.

The light and heavy chain variable regions are responsible for binding the target antigen and can therefore show significant sequence diversity between antibodies. The constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events. The variable region of an antibody contains the antigen-binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The majority of sequence variability occurs in six hypervariable regions, three each per variable heavy (V_(H)) and light (V_(L)) chain; the hypervariable regions combine to form the antigen-binding site, and contribute to binding and recognition of an antigenic determinant. The specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions, as well as their size, shape, and chemistry of the surface they present to the antigen. Various schemes exist for identification of the regions of hypervariability, the two most common being those of Kabat and of Chothia and Lesk. Kabat et al (1991) define the “complementarity-determining regions” (CDR) based on sequence variability at the antigen-binding regions of the V_(H) and V_(L) domains. Chothia and Lesk (1987) define the “hypervariable loops” (H or L) based on the location of the structural loop regions in the V_(H) and V_(L) domains. These individual schemes define CDR and hypervariable loop regions that are adjacent or overlapping, those of skill in the antibody art often utilize the terms “CDR” and “hypervariable loop” interchangeably, and they may be so used herein. The CDR/loops are identified herein according to the Kabat scheme.

An “antibody fragment” as referred to herein may include any suitable antigen-binding antibody fragment known in the art. The antibody fragment may be a naturally-occurring antibody fragment, or may be obtained by manipulation of a naturally-occurring antibody or by using recombinant methods. For example, an antibody fragment may include, but is not limited to a Fv, single-chain Fv (scFv; a molecule consisting of V_(L) and V_(H) connected with a peptide linker), Fab, F(ab′)₂, single-domain antibody (sdAb; a fragment composed of a single V_(L) or V_(H)), and multivalent presentations of any of these. Antibody fragments such as those just described may require linker sequences, disulfide bonds, or other type of covalent bond to link different portions of the fragments; those of skill in the art will be familiar with the requirements of the different types of fragments and various approaches and various approaches for their construction.

In a non-limiting example, the antibody fragment may be an sdAb derived from naturally-occurring sources. Heavy chain antibodies of camelid origin (Hamers-Casterman et al, 1993) lack light chains and thus their antigen binding sites consist of one domain, termed V_(H)H. sdAb have also been observed in shark and are termed V_(NAR) (Nuttall et al, 2003). Other sdAb may be engineered based on human Ig heavy and light chain sequences (Jespers et al, 2004; To et al, 2005). As used herein, the term “sdAb” includes those sdAb directly isolated from V_(H), V_(H)H, V_(L), or V_(NAR) reservoir of any origin through phage display or other technologies, sdAb derived from the aforementioned sdAb, recombinantly produced sdAb, as well as those sdAb generated through further modification of such sdAb by humanization, affinity maturation, stabilization, solubilization, camelization, or other methods of antibody engineering. Also encompassed by the present invention are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the sdAb.

SdAb possess desirable properties for antibody molecules, such as high thermostability, high detergent resistance, relatively high resistance to proteases (Dumoulin et al, 2002) and high production yield (Arbabi-Ghahroudi et al, 1997); they can also be engineered to have very high affinity by isolation from an immune library (Li et al, 2009) or by in vitro affinity maturation (Davies & Riechmann, 1996). Further modifications to increase stability, such as the introduction of non-canonical disulfide bonds (Hussack et al, 2011a,b; Kim et al, 2012), may also be brought to the sdAb.

A person of skill in the art would be well-acquainted with the structure of a single-domain antibody (see, for example, 3DWT, 2P42 in Protein Data Bank). An sdAb comprises a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDR/hypervariable loops form the antigen-binding site. However, and as would be understood by those of skill in the art, not all CDR may be required for binding the antigen. For example, and without wishing to be limiting, one, two, or three of the CDR may contribute to binding and recognition of the antigen by the sdAb of the present invention. The CDR of the sdAb or variable domain are referred to herein as CDR1, CDR2, and CDR3.

The antibody or fragment thereof as described herein may transmigrate the blood-brain barrier. The brain is separated from the rest of the body by a specialized endothelial tissue known as the blood-brain barrier (BBB). The endothelial cells of the BBB are connected by tight junctions and efficiently prevent many therapeutic compounds from entering the brain. In addition to low rates of vesicular transport, one specific feature of the BBB is the existence of enzymatic barrier(s) and high level(s) of expression of ATP-dependent transporters on the abluminal (brain) side of the BBB, including P-glycoprotein (Gottesman and Pastani, 1993; Watanabe, 1995), which actively transport various molecules from the brain into the blood stream (Samuels, 1993). Only small (<500 Daltons) and hydrophobic (Pardridge, 1995) molecules can more readily cross the BBB. Thus, the ability of the antibody or fragment thereof as described above to specifically bind the surface receptor, internalize into brain endothelial cells, and undergo transcytosis across the blood brain barrier by evading lysosomal degradation is useful in the neurological field. The antibody or fragment thereof that crosses the blood-brain barrier may be used to carry other molecules, such as therapeutics, for delivery to the brain tissue. The antibody or fragment thereof may be any suitable antibody or fragment thereof known in the art to transmigrate the blood brain barrier.

The present invention provides a compound, or fusion protein, comprising an antibody or fragment thereof that transmigrates the blood brain barrier (BBB). An antibody or fragment of the present invention may bind to, for example, transmembrane protein 30A (TMEM30A), as described in WO 2007/036021, or to an Insulin-Like Growth Factor 1 Receptor (IGF1R) epitope, or isoforms, variants, portions, or fragments thereof.

The antibody or fragment thereof in the compound of the present invention may comprise a complementarity determining region (CDR) 1 sequence of HYTMG (SEQ ID NO:1); a CDR2 sequence of RITWGGDNTFYSNSVKG (SEQ ID NO:2); and a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:3); or

-   -   a CDR1 sequence of EYPSNFYA (SEQ ID NO:4), a CDR2 sequence of         VSRDGLTT (SEQ ID NO:5), a CDR3 sequence of AIVITGVWNKVDVNSRSYHY         (SEQ ID NO:6); or     -   a CDR1 sequence of GGTVSPTA (SEQ ID NO:7), a CDR2 sequence of         ITWSRGTT (SEQ ID NO:8), a CDR3 sequence of AASTFLRILPEESAYTY         (SEQ ID NO:9); or     -   a CDR1 sequence of GRTIDNYA (SEQ ID NO:10), a CDR2 sequence of         IDWGDGGX; where X is A or T (SEQ ID NO:11), a CDR3 sequence of         AMARQSRVNLDVARYDY (SEQ ID NO:12).

As previously stated, the antibody or fragment thereof may be an sdAb of camelid origin or derived from a camelid V_(H)H, and thus may be based on camelid framework regions; alternatively, the CDR described above may be grafted onto V_(NAR), V_(H)H, V_(H) or V_(L) framework regions. In yet another alternative, the hypervariable loops described above may be grafted onto the framework regions of other types of antibody fragments (Fv, scFv, Fab) of any source (for example, mouse or human) or proteins of similar size and nature onto which CDR can be grafted (for example, see Nicaise et al, 2004).

The present invention further encompasses an antibody or fragment thereof that is chimeric (or chimerized), veneered, or humanized. Chimeric antibodies or fragments thereof are constructs in which the native variable domain (of mouse or camelid origin) is linked to human constant domain(s) (see Gonzales et al 2005). Veneering or re-surfacing of antibodies involves replacing exposed residues in the framework region of the native antibody or fragment thereof with the amino acid residues in their human counterpart (Padlan, 1991; Gonzales et al 2005). Humanization of an antibody or antibody fragment comprises replacing an amino acid in the sequence with its human counterpart, as found in the human consensus sequence, without loss of antigen-binding ability or specificity; this approach reduces immunogenicity of the antibody or fragment thereof when introduced into human subjects. In this process, one or more than one of the CDR defined herein may be fused or grafted to a human variable region (V_(H), or V_(L)), to other human antibody (IgA, IgD, IgE, IgG, and IgM), to human antibody fragment framework regions (Fv, scFv, Fab), or to human proteins of similar size and nature onto which CDR can be grafted (Nicaise et al, 2004). In such a case, the conformation of said one or more than one hypervariable loop is likely preserved, and the affinity and specificity of the sdAb for its target (i.e., an epitope on brain endothelial cells, such as TMEM30A, or an IGF1R epitope) brain endothelial cells) is likely minimally affected. As is known by those of skill in the art, it may be necessary to incorporate certain native amino acid residues into the human framework in order to retain binding and specificity. Humanization by CDR grafting is known in the art (for example, see Tsurushita et al, 2005; Jones et al, 1986; Tempest et al, 1991; Riechmann et al, 1988; Queen et al, 1989; reviewed in Gonzales et al, 2005—see also references cited therein), and thus persons of skill would be amply familiar with methods of preparing such humanized antibody or fragments thereof.

The provided antibody or fragment thereof may be a humanized version of the FC5 antibody (described in WO 2002/057445) or an IGF1R antibody. FC5 (comprising a sequence of any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17), and IGF1R (comprising a sequence of any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26) bind to the surface of receptor epitopes on brain endothelial cells and subsequently transmigrates the blood-brain barrier (BBB). FC5 has also been shown to act as a carrier to usher molecules of various sizes across the BBB (see for example, WO 2011/127580). The antigen mediating FC5 transmigration was tentatively identified as transmembrane domain protein 30A (TMEM30A; WO 2007/036021), which is enriched on the surface of brain endothelial cells.

For example, and without wishing to be limiting, the antibody or fragment thereof may comprise the sequence:

(SEQ ID NO: 13) X₁VQLVX₂SGGGLVQPGGSLRLSCAASGFKITHYT MGWX₃RQAPGKX₄X₅EX₆VSRITWGGDNTFYSNSV KGRFTISRDNSKNTX₇YLQMNSLRAEDTAVYYCAA GSTSTATPLRVDYWGQGTLVTVSS, where X₁=D or E, X₂=A or E, X₃=F or V, X₄=E or G, X₅=R or L, X₆=F or W, X₇=L or V, or a sequence substantially identical thereto;

(SEQ ID NO: 18) X₁VX₂LX₃ESGGGLVQX₄GGSLRLSCX₅ASEYPSN FYAMSWX₆RQAPGKX₇X₈EX₉VX₁₀GVSRDGLTTL YADSVKGRFTX₁₁SRDNX₁₂KNTX₁₃X₁₄LQMNSX₁₅ X₁₆AEDTAVYYCAIVITGVWNKVDVNSRSYHYW GQGTX₁₇VTVSS, where X₁ is E or Q; X₂ is K or Q; X₃ is V or E; X₄ is A or P; X₅ is V or A; X₆ is F or V; X₇ is E or G; X₈ is R or L; X₉ is F or W; X₁₀ is A or S; X₁₁ is M or I; X₁₂ is A or S; X₁₃ is V or L; X₁₄ is D or Y; X₁₅ is V or L; X₁₆ is K or R; and X₁₇ is Q or L; or a sequence substantially identical thereto;

(SEQ ID NO: 21) X₁VX₂LX₃ESGGGLVQX₄GGSLRLSCX₅X₆SGGTV SPTAMGWX₇RQAPGKX₈X₉EX₁₀VX₁₁HITWSRGT TRX₁₂ASSVKX₁₃RFTISRDX₁₄X₁₅KNTX₁₆YLQ MNSLX₁₇X₁₈EDTAVYYCAASTFLRILPEESAY TYWGQGT X₁₉VTVSS, where X₁ is E or Q; X₂ is K or Q; X₃ is V or E; X₄ is A or P; X₅ is A or E; X₆ is V or A; X₇ is V or F; X₈ is G or E; X₉ is L or R; X₁₀ is F or W; X₁₁ is G or S; X₁₂ is V or Y; X₁₃ is D or G; X₁₄ is N or S; X₁₅ is A or S; X₁₆ is L or V; X₁₇ is K or R; X₁ is A or S; and X₁₉ is L or Q; or a sequence substantially identical thereto;

(SEQ ID NO: 24) X₁VX₂LX₃ESGGGLVQX₄GGSLRLSCAASGRTIDN YAMAWX₅RQAPGKX₆X₇EX₈VX₉TIDWGDGGX₁₀R YANSVKGRFTISRDNX₁₁KX₁₂TX₁₃YLQMNX₁₄L X₁₅X₁₆EDTAVYX₁₇CAMARQSRVNLDVARYDYWG QGTX₁₈VTVSS, where X₁ is E or Q; X₂ is K or Q; X₃ is V or E; X₄ is A or P; X₅ is V or S; X₆ is D or G; X₇ is L or R; X₈ is F or W; X₉ is A or S; X₁₀ is A or T; X₁₁ is A or S; X₁₂ is G or N; X₁₃ is M or L; X₁₄ is N or R; X₁₅ is E or R; X₁₆ is P or A; X₁₇ is S or Y; and X₁₈ is Q or L; or a sequence substantially identical thereto.

More specifically, and without wishing to be limiting in any manner, the antibody or fragment thereof may comprise a sequence selected from any one of:

(SEQ ID NO: 14) DVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMG WFRQAPGKEREFVSRITWGGDNTFYSNSVKGRFTI SRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTAT PLRVDYWGKGTQVTVSS; (SEQ ID NO: 15) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMG WVRQAPGKGLEWVSRITWGGDNTFYSNSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAAGSTSTAT PLRVDYWGQGTLVTVSS; (SEQ ID NO: 16) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMG WVRQAPGKGLEWVSRITWGGDNTFYSNSVKGRFTI SRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTAT PLRVDYWGQGTLVTVSS; (SEQ ID NO: 17) EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMG WFRQAPGKGLEFVSRITWGGDNTFYSNSVKGRFTI SRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTAT PLRVDYWGQGTLVTVSS; (SEQ ID NO: 19) QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMS WFRQAPGKEREFVAGVSRDGLTTLYADSVKGRFTM SRDNAKNTVDLQMNSVKAEDTAVYYCAIVITGVWN KVDVNSRSYHYWGQGTQVTVSS; (SEQ ID NO: 20) EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMS WFRQAPGKEREFVSGVSRDGLTTLYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWN KVDVNSRSYHYWGQGTLVTVSS; (SEQ ID NO: 22) QVKLEESGGGLVQAGGSLRLSCEVSGGTVSPTAMG WFRQAPGKEREFVGHITWSRGTTRVASSVKDRFTI SRDSAKNTVYLQMNSLKSEDTAVYYCAASTFLRIL PEESAYTYWGQGTQVTVSS; (SEQ ID NO: 23) QVQLVESGGGLVQPGGSLRLSCAVSGGTVSPTAMG WFRQAPGKGLEFVGHITWSRGTTRYASSVKGRFTI SRDNSKNTVYLQMNSLRAEDTAVYYCAASTFLRIL PEESAYTYWGQGTLVTVSS; (SEQ ID NO: 25) QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMA WSRQAPGKDREFVATIDWGDGGARYANSVKGRFTI SRDNAKGTMYLQMNNLEPEDTAVYSCAMARQSRVN LDVARYDYWGQGTQVTVSS; (SEQ ID NO: 26) QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMA WVRQAPGKGLEWVATIDWGDGGTRYANSVKGRFTI SRDNSKNTMYLQMNSLRAEDTAVYYCAMARQSRVN LDVARYDYWGQGTLVTVSS; and a sequence substantially identical thereto. The antibody or fragment thereof may be a single-domain antibody.

With respect to any component peptide in the fusion protein of the present invention, a “substantially identical” sequence may comprise one or more conservative amino acid mutations, or amino acid deletions that allow for biologically functional activity to be maintained (such as in the C-terminally cleaved ABP peptides provided herein). It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, physico-chemical or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered “substantially identical” polypeptides. A conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity). These conservative amino acid mutations may be made to the framework regions of the sdAb while maintaining the CDR sequences listed above and the overall structure of the CDR of the antibody or fragment; thus the specificity and binding of the antibody are maintained.

With respect to any component peptide in the fusion protein of the present invention, a “substantially equivalent” sequence may comprise one or more conservative amino acid mutations or amino acid deletions that allow for biological functional activity to be maintained (such as in the C-terminal cleaved ABP peptides provided herein); wherein the mutant peptide is substantially equivalent with respect to peptide stability and bio-manufacturability. Substantially equivalent may refer to equivalent with respect to fusion molecule stability; for example, the lack of a degradation product, or a low molecular weight band, as seen in SDS PAGE (reducing and non-reducing conditions). It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, physico-chemical or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered “substantially equivalent” polypeptides.

In a non-limiting example, a conservative mutation may be an amino acid substitution or deletions that maintain functionality. Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term “basic amino acid” it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term “neutral amino acid” (also “polar amino acid”), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term “hydrophobic amino acid” (also “non-polar amino acid”) is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (lie or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). “Acidic amino acid” refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).

Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or any other appropriate software that is known in the art.

The substantially identical sequences of the present invention may be at least 74% identical; in another example, the substantially identical sequences may be at least 74, 75, 76, 77, 78, 79, 80-90, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical, or any percentage therebetween, at the amino acid level to sequences described herein. Importantly, the substantially identical sequences retain the activity and specificity of the reference sequence. In a non-limiting embodiment, the difference in sequence identity may be due to conservative amino acid mutation(s). In a non-limiting example, the present invention may be directed to an antibody or fragment thereof comprising a sequence at least 95%, 98%, or 99% identical to that of the antibodies described herein. In another non-limiting example, as provided herein, the ABP peptide of the present invention may comprise a sequence that is at least 74% identical to SEQ ID NO: 36 (40 aa); that is to say that the present invention comprises an ABP peptide that is a C-terminal cleavage product of SEQ ID NO: 36 and may be at least 74%, 75%, 76%, 77%, 78%-100% identical thereto, or any percentage therebetween. It should be noted that percent identity is calculated based on each component peptide relative to the reference peptide sequence (i.e. comparing ABP component peptide sequences). An ABP of the present invention comprises a sequence of SEQ ID NO: 46 (29 aa), or an equivalent β-amyloid binding protein thereof.

The antibody or fragment thereof in the compound of the present invention may be linked to an Fc domain, for example, but not limited to human Fc domains. The Fc domains may be selected from various classes including, but not limited to, IgG, IgM, or various subclasses including, but not limited to IgG1, IgG2, etc. In this approach, the Fc gene is inserted into a vector along with the sdAb gene to generate a sdAb-Fc fusion protein (Bell et al, 2010; Iqbal et al, 2010); the fusion protein is recombinantly expressed then purified. For example, and without wishing to be limiting in any manner, multivalent display formats may encompass chimeric formats of FC5-H3 and its mutational variants linked to an Fc domain. Such antibodies are easy to engineer and to produce, can greatly extend the serum half-life of sdAb (Bell et al., 2010).

The Fc domain in the compound as just described may be any suitable Fc fragment known in the art. The Fc fragment may be from any suitable source; for example, the Fc may be of mouse or human origin. Other Fc or Fc fragment embodiments may modulate, modify or suppress an immunological effector function (Shields 2001). Other Fc fragment embodiments may mediate clearance of the fusion peptide from the brain (Caram-Salas N 2011). In a specific, non-limiting example, the Fc may be the mouse Fc2a fragment or human Fc1 fragment (Bell et al, 2010; Iqbal et al, 2010). In a specific, non-limiting example, the multimerized construct may comprise the isolated or purified antibody or fragment as described herein and an Fc of sequence of SEQ ID NO:48; SEQ ID NO:49; SEQ ID NO:50. Accordingly, the BBB-Fc-ABP fusion proteins provided herein may form dimers via the Fc to provide a bivalent, bifunctional BBB-Fc-ABP.

The compound of the present invention comprises an antibody or fragment thereof linked to a polypeptide that binds β-amyloid. The linker may be any polypeptide, comprising neutral or hydrophilic amino acids, of suitable length. In a non-limiting example, the length is preferably less than 12 amino acids, and the polypeptide is GGGGSGGGGS, or TGGGGSGGGGS, or any suitable linker (ex. (GGGSGGGGS)n or (GGGGSGGG)n or any suitable combination). Other chemical linkers may be employed using non-peptidic forms such as amide or ester linkages in a correct orientation. For example, SEQ ID NO: 71 provides a fusion protein comprising any suitable linker, wherein any fusion protein of the present invention, i.e. SEQ ID NO: 51-70 may comprise any suitable linker as exemplified in SEQ ID NO: 71. The polypeptide that binds β-amyloid (Aβ) may bind pathologically relevant β-amyloids, such as Aβ₁₋₄₂ aggregates, which are implicated in AD pathology; the polypeptide may bind Aβ with high affinity (in the nM range), inhibit Aβ binding to cellular proteins and Aβ₁₋₄₂-induced cell toxicity in vitro, and binds amyloid deposits in AD transgenic mice brain as well as in the brains from AD patients in vitro. The polypeptide in the compound of the present invention does not bind the reverse peptide Aβ₄₂₋₁.

In a compound provided herein, the polypeptide that binds β-amyloid may comprise a sequence selected from the group consisting of: SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:35; SEQ ID NO:36 or a β-amyloid binding C-terminal cleavage product thereof, namely SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43; or SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46, SEQ ID NO:47 and a sequence substantially identical thereto. A “substantially identical” sequence is as described above.

Accordingly, the present invention provides a polypeptide that binds β-amyloid and variants thereof (i.e. ABP and ABP variants). The ABP variant may comprise a sequence having SEQ ID NO: 31, for example, the ABP variant provided may comprise a sequence selected from the group consisting of: SEQ ID NO: 31 to SEQ ID NO:47 or a sequence substantially equivalent thereto.

Accordingly, there is provided ABP polypeptide sequences comprising specific systematic and methodical modifications based on detailed biophysical characterization of the ABP. The specific and methodically directed modifications to the ABP polypeptide comprise the novel and unobvious ABP variants of the present invention, as provided in SEQ ID NO:31, or a C-terminal cleavage product having β-amyloid binding activity.

The peptide provided herein may comprise an ABP comprising a sequence that may be selected from the group consisting of SEQ ID NO: 27-SEQ ID NO: 47, and a sequence substantially equivalent thereto or a C-terminal cleavage product having β-amyloid binding activity.

The fusion protein provided herein may comprise an ABP comprising a sequence that may be selected from any one of SEQ ID NO: 31 to SEQ ID NO:47. The ABP may be any equivalent sequence to SEQ ID NO:31 or a C-terminal cleavage product of SEQ ID NO: 31 or 36 wherein said C-terminal product has β-amyloid binding activity, such as SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43. The ABP may be any sequence equivalent to SEQ ID NO: 46. A construct comprising an ABP, an ABP variant or a C-terminal cleavage product, as provided herein, exhibits advantageous improvement in compound stability and bio-manufacturability (as can be seen in FIG. 16 ).

More specifically, the specific ABP cleavage species provided herein (namely SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43) could not have been predicted, and the now specified cleavage sites C-terminally cleaved from SEQ ID NO: 36 were not known and would not have been predicted. These specific C-terminal cleavage products could not have been predicted during biomanufacturing of the fusion peptides. The stability and biomanufacturability of the ABP proteins and the C-terminal cleavage products thereof could not have been predicted.

Furthermore, it is quite advantageous and unexpected that the fusion proteins provided herein can be bifunctional dimers or monofunctional dimers, wherein the monofunctional and bifunctional dimers provide equivalent fusion proteins with respect to β-amyloid binding functionality and activity. The advantageous stability, manufacturability and equivalent β-amyloid binding functionality of the provided C-terminal cleavage products was not known and not obvious. This allows for the manufacture of a mixture of ABP peptides having equivalent β-amyloid binding activity, and for the manufacture of a mixture of fusion proteins and dimers thereof having equivalent β-amyloid binding activity. Moreover, the advantageous equivalent functionality of monofunctional and bifunctional dimers was not known and could not have been predicted. It is quite advantageous that the present invention provides a mixture of fusion proteins and dimers (monofunctional and bifunctional dimers comprising a minimum ABP sequence comprising SEQ ID NO: 46) of equivalent activity, thereby allowing for an increase in functional ABP protein yield and an increase in functional fusion protein and functional fusion protein dimer yield in biomanufacturing.

The fusion proteins and compounds provided herein, exhibit improved therapeutic efficacy. More specifically, the compounds provided comprise specifically modified ABP, which includes, for example mutations of specific amino acids K (Lysine), R (Arginine) and N (Aspargine) to G (Glycine) at specific sites—position 1, 4, 6, 7, 28 and 29 from the N-terminus of ABP sequence (SEQ ID NO: 36), that allows the generation of stable BBB-Fc-ABP fusion molecule for enhanced bio-manufacturability (production in human mammalian expression system). The specific modifications to ABP provided herein were systematic and methodical modifications based on detailed biophysical characterization of the ABP. The modified ABP provided herein, may comprise, for example, a sequence selected from SEQ ID NO: 32-SEQ ID NO: 47, and a sequence substantially equivalent thereto, such as SEQ ID NO: 31, or a C-terminally cleaved functional product thereof. The compounds provided herein advantageously exhibit improved stability and bio-manufacturability, and a most significant increase in the efficacy of reducing Aβ levels in brain; wherein a 50% amyloid reduction was observed within 24 hr of treatment with a construct of the present invention.

By the term “linked”, also referred to herein as “conjugated”, it is meant that two moieties are joined directly or indirectly (e.g., via a linker), covalently or non-covalently (e.g., adsorption, ionic interaction). A covalent linkage may be achieved through a chemical cross-linking reaction, or through fusion using recombinant DNA methodology combined with any peptide expression system, such as bacteria, yeast or mammalian cell-based systems. When conjugating the antibody or fragment thereof to the polypeptide binding Aβ or Fc, a suitable linker may be used. For example, a suitable linker may be any polypeptide, comprising neutral or hydrophilic amino acids, of suitable length that allows for the conjugation of the components of the BBB-Fc-ABP protein fusion. For example, the linker that allows the components of the fusion protein (for example, in SEQ ID NO: 51-69) to be according linked, and is not limited to the GGGGSGGGGS or (GGGS)_(n) linker highlighted therein and may be any suitable linker (i.e. as in the non-limited fusion protein of SEQ ID NO: 71). In a non-limiting example, the length is preferably less than 12 amino acids, and the polypeptide may be GGGGSGGGS, or GGGSGGGGS, or TGGGGSGGGS, or any suitable linker in the art. Other chemical linkers may be employed using non-peptidic forms such as amide or ester linkages in a correct orientation. One of skill in the present art would be well aware of linkers or method of linking an antibody or fragment thereof to a polypeptide. Methods for linking an antibody or fragment thereof to a polypeptide or Fc are well-known to a person of skill in the art.

The compound provided herein comprises an antibody or fragment thereof, a polypeptide that binds β-amyloid, and an Fc fragment, linked to provide a construct (also referred to herein as a compound or fusion molecule), wherein the construct comprises a fusion protein and dimers thereof. The antibody or fragment thereof may be linked to a polypeptide that binds β-amyloid via an Fc fragment, or a suitable linker.

The antibody or fragment thereof comprises a sequence selected from any one of: SEQ ID NO:14, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO 23, SEQ ID NO:25, SEQ ID NO:26, and a sequence substantially identical thereto. The antibody or fragment thereof transmigrates the blood brain barrier. The antibody or fragment may be a sdAb; wherein the sdAb may be humanized.

The polypeptide that binds β-amyloid comprises a sequence selected from the group consisting of: SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and a sequence substantially identical thereto, and a sequence comprising or consisting of SEQ ID NO: 31 or a C-terminal cleavage product having β-amyloid binding activity, or a sequence comprising or consisting of SEQ ID NO: 46.

The Fc fragment comprises a sequence selected from any one of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and a sequence substantially identical thereto. The Fc fragment of the present invention may be any suitable Fc fragment with attenuated effector function. The Fc fragment provided in the fusion protein allows for the formation of dimeric structures; wherein for example a single-chain fusion protein comprising a sequence selected from the group consisting of SEQ ID NO: 51 to SEQ ID NO: 71 may form dimeric structures conjugated via the Fc fragment therein.

Accordingly, a compound or construct of the present invention may comprise a sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, and a sequence substantially identical thereto, or dimeric structures thereof, wherein the dimeric peptide comprising two single chain polypeptides selected from the group comprising SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 SEQ ID NO: 70, SEQ ID NO: 71, and a sequence substantially identical thereto, a deletion variant such as SEQ ID NO: 47 or any C-terminally cleaved ABP variant, sequential or otherwise, derived from SEQ ID NO; 31, consisting of a functional product comprising SEQ ID NO: 46; specifically, there is provided sequential one amino acid C-terminal deletions of SEQ ID NO 31 or minus 1, 2, 3, 4, 5, 6, 7, or 8 amino acids (as provided in SEQ ID NO: 37 to 43). The single chain polypeptides of the dimer may be bifunctional homodimers (FIG. 1Ai), bifunctional heterodimers (FIG. 1Aii) or monofunctional heterodimers (FIG. 1Aiii). For example, the ABP portion of each dimer may be the same or different; when the ABP portion is different in the dimer, at least one of the ABP in the single chain polypeptide is a biologically functional peptide (i.e. capable of binding β-amyloid).

The dimeric peptide comprising two BBB-Fc-L-ABP single-chain polypeptides may comprise two different ABP sequences (heterodimer), wherein at least one of said ABP sequences in the heterodimer is a biologically functional ABP molecule (i.e. can bind β-amyloid, as represented in FIG. 1Aii or 1Aiii) as described in FIGS. 23A-23C, 24A-24B, and 25 . It is quite advantageous that the dimeric peptide may accommodate a single non-functional ABP peptide (i.e. monofunctional heterodimer as represented in FIG. 1Aiii) and maintain an equivalent functional biological activity with respect to β-amyloid binding (FIGS. 23B-23C). In a dimer comprising two different ABP sequences (i.e. heterodimeric with respect to ABP), at least one of the ABP peptides provided in the heterodimer is a biologically functional ABP i.e. SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 or any combination thereof when two functional ABP peptides are provided in the dimer (bifunctional heterodimer). It is quite advantageous with respect to manufacturability, isolability and functional product yield, that it is not necessary that both ABP peptides in the heterodimer be biologically functional ß-amyloid binding proteins, having monofunctional (or bifunctional) heterodimers maintains the overall biological activity of the fusion protein with respect to β-amyloid binding activity in vitro (FIGS. 23A-23B) and in vivo brain uptake and efficacy (FIGS. 24A-24B and 25 ). The ß-amyloid binding activity (EC 50) of the fractions containing a mixture of mono- and bi-functional homo- and hetero-dimers (PEAK 2, FIG. 23A) was very similar to that of the bifunctional homo- and hetero-dimer (PEAK 3, FIG. 23A allowing the mono- and bi-functional hetero- and homo-dimer fractions (PEAKS 2 and 3 of the CEX) to be combined. It is advantageous that a heterodimer can accommodate a single non-functional ABP peptide (monofunctional) and remain a biologically effective heterodimer as it aids in the manufacturability of the provided fusion protein. In a specific example, combining homodimer and heterodimer pools during down-stream processing (purification) of FC5(H3)-hFc1x7-ABP fusion protein (SEQ ID NO: 56), the product yield increased significantly, from ˜65% to ˜86% (see FIGS. 23A-23C), during large-scale biomanufacturing, this is a very distinct advantage. The advantages provided by the unexpected maintenance of equivalent functionality of the monofunctional heterodimers increases the product yield and facilitates the manufacturability, permitting the ready isolation of fully-active pharmaceutical compositions (increasing fusion protein yield, which allows for an increased yield in the functional fusion protein dimers produced). Having monofunctional heterodimers that are equally active to their corresponding bifunctional dimers allows for the increased yield of the manufactured fusion protein, and thereby facilitates and increases the manufacturability of a composition comprising a mixture of the fusion proteins and their corresponding dimers.

The present invention provides fusion proteins comprising a BBB-crossing single-domain antibody (BBB), an Fc fragment (Fc) and an amyloid-binding peptide (ABP), wherein each moiety may be linked to provide fusion molecules, as illustrated in FIGS. 1Ai-1D. For example, in a non-limiting embodiment of the represent invention, the BBB may be linked to the N-terminus of the Fc and the ABP linked to the C-terminus of the Fc fragment (FIG. 1Ai-1Aiii). FIGS. 1Ai-1D show the corresponding dimers of the single-chain fusion protein comprising BBB-Fc-ABP. The dimer of the fusion protein could be a homodimer with respect to ABP in that both ABP arms contain functional full-length (e.g., a bifunctional homodimer, ex SEQ ID No: 36, FIG. 1Ai) and/or various C-terminally cleaved ABPs (e.g., a bifunctional heterodimer SEQ ID No: 36 and/or SEQ ID No: 37 to SEQ ID No: 43, FIG. 1Aii,) or a heterodimer wherein one of the ABP arms is functional (ABP or C-terminally cleaved) and the other arm is non-functional (i.e. a monofunctional heterodimer). The 3 components (i.e. BBB, Fc, and ABP) are depicted in various configurations, as illustrated in FIGS. 1Ai-1Aiii, 1B, 1C, and 1D. In a specific, non-limiting example of the compound of the present invention, the polypeptide that binds β-amyloid may comprise the sequence SEQ ID NO: 31 or a C-terminal cleaved functional product thereof.

In an embodiment the ABP variant comprises the sequence:

GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI (SEQ ID NO:36) referred to herein as ABP(6G) and a sequence substantially equivalent thereto, or any C-terminal cleavage product having β-amyloid binding activity, such as SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43. In a embodiment, the ABP comprises a sequence of SEQ ID NO: 46.

The antibody or fragment thereof may comprise the sequence

-   -   EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSRITWGGD         NTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG         QGTLVTVSS (SEQ ID NO:17) referred to herein as FC5-H3.

The antibody or fragment thereof may further comprise the sequence of a human Fc, such as

-   -   AEPKSSDKTHTCPPCPAPEILGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPE         VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL         PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ         PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG         (SEQ ID NO:49), also referred to herein as hFc1X7.

Without wishing to be limiting in any manner, the compound of the present invention may comprise the sequence:

-   -   EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEFVSRITWGGD         NTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGSTSTATPLRVDYWG         QGTLVTVSSAEPKSSDKTHTCPPCPAPEILGGPSVFLFPPKPKDTLMISRTPEVTCVV         VDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY         KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA         VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN         HYTQKSLSLSPGTGGGGSGGGGSGTFGTGGASAQASLASKDKTPKSKSKKGGSTQL KSRVKNI         [SEQ ID NO:56, also referred to herein as         FC5-H3-hFc1X7-ABP(6G)]. The present invention also provides a         fusion protein of SEQ ID NO: 56 wherein the linker linking the         Fc and ABP components of the fusion is (GGGGS)2, (GGGS)2,         T(GGGGS)2 or any suitable linker known in the art, and wherein         the ABP peptide of the fusion protein of SEQ ID NO: 56 may be         SEQ ID NO:36, a sequence substantially equivalent thereto, or         any C-terminal cleavage product having functional β-amyloid         binding activity, such as SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID         NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:         43 or SEQ ID NO: 47 (SEQ ID NOs: 57-63, SEQ ID NO: 65).

TABLE Summary of some representative BBB-Fc-ABP fusion protein constructs. BBB Fc ABP FC5 (SEQ ID NO: 14) mFc2a (SEQ ID NO: 48) ABP (SEQ ID NO: 30) FC5(H3) (SEQ ID NO: 17) mFc2a (SEQ ID NO: 48) ABP (SEQ ID NO: 30) FC5(H3) (SEQ ID NO: 17) hFc1x7 (SEQ ID NO: 49) ABP (SEQ ID NO: 30) FC5(H3) (SEQ ID NO: 17) hFc1x7 (SEQ ID NO: 49) ABP(G) SEQ ID NO: 32) FC5(H3) (SEQ ID NO: 17) hFc1x7 (SEQ ID NO: 49) ABP(GG-G) SEQ ID NO: 35 FC5(H3) (SEQ ID NO: 17) hFc1x7 (SEQ ID NO: 49) ABP(6G) SEQ ID NO: 36, SED ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43) FC5(H1) (SEQ ID NO: 15) hFc1x7 (SEQ ID NO: 49) ABP(6G) SEQ ID NO: 36, SED ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43) FC5(H2) (SEQ ID NO: 16) hFc1x7 (SEQ ID NO: 49) ABP(6G) SEQ ID NO: 36, SED ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43) FC5(H3) (SEQ ID NO: 17) hFc1X0 (SEQ ID No: 50) ABP(GG-G) SEQ ID NO: 35 FC5(H3) (SEQ ID NO: 17) hFc1x7 (SEQ ID NO: 49) ABP(trc) SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47) IGF1R-5(H2) (SEQ ID NO: 26) mFc2a (SEQ ID NO: 48) ABP (SEQ ID NO: 30)

It is noted that in the above-table, the BBB-Fc-ABP fusion protein constructs may also comprise a linker (L) wherein the BBB-Fc-ABP construct is a BBB-Fc-L-ABP fusion protein of the present invention, wherein L may be GGGGSGGGGS, GGGSGGGGS, TGGGGSGGGGS or any multiple thereof, or any suitable linker, or any linker as exemplified in the consensus linker sequence in SEQ ID NO:53.

The compound of the present invention, as provided in the fusion protein of SEQ ID NO:56 also referred to herein as FC5-H3-hFc1X7-L-ABP(6G), may comprise variations in each of the components comprised therein, for example, ABP may be ABP (GG-G), as provided in SEQ ID NO: 55 or ABP(6G) SEQ ID NO: 36, or any C-terminal cleavage product thereof. The linker sequence provided therein (as highlighted for example, in SEQ ID NO:51 to SEQ ID NO: 71) may be any linker that allows for the linking of the BBB-Fc-ABP fusion protein. The compound of the present invention may also comprise additional sequences to aid in expression, detection or purification of a recombinant antibody or fragment thereof. Any such sequences or tags known to those of skill in the art may be used. For example, and without wishing to be limiting, the antibody or fragment thereof may comprise a targeting or signal sequence (for example, but not limited to ompA), a detection/purification tag (for example, but not limited to c-Myc, His₅, or His₆), or a combination thereof. In another example, the additional sequence may be a biotin recognition site such as that described by Cronan et al (WO 95/04069) or Voges et al (WO/2004/076670). As is also known to those of skill in the art, linker sequences may be used in conjunction with the additional sequences or tags, or may serve as a detection/purification tag.

The present invention also encompasses nucleic acid sequences encoding the compounds as described herein. Given the degeneracy of the genetic code, a number of nucleotide sequences would have the effect of encoding the polypeptide, as would be readily understood by a skilled artisan. The nucleic acid sequence may be codon-optimized for expression in various micro-organisms. The present invention also encompasses vectors comprising the nucleic acids as just described. Furthermore, the invention encompasses cells comprising the nucleic acid and/or vector as described.

The present invention further encompasses a composition comprising one or more than one compound as described herein and a pharmaceutically acceptable diluent, excipient, or carrier. The composition may also comprise a pharmaceutically acceptable diluent, excipient, or carrier. The diluent, excipient, or carrier may be any suitable diluent, excipient, or carrier known in the art, and must be compatible with other ingredients in the composition, with the method of delivery of the composition, and is not deleterious to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in suspension form or powder form (for example, but limited to lyophilised or encapsulated). For example, and without wishing to be limiting, when the composition is provided in suspension form, the carrier may comprise water, saline, a suitable buffer, or additives to improve solubility and/or stability; reconstitution to produce the suspension is effected in a buffer at a suitable pH to ensure the viability of the antibody or fragment thereof. Dry powders may also include additives to improve stability and/or carriers to increase bulk/volume; for example, and without wishing to be limiting, the dry powder composition may comprise sucrose or trehalose. It would be within the competency of a person of skill in the art to prepare suitable compositions comprising the present compounds.

A method of treating Alzheimer's disease is also provided, in which a compound or composition of the present invention is administered to a subject in need thereof. Any appropriate route of administration may be utilized, including but not limited to intravenous, intraperitoneal, parenteral, intracranial, intramuscular, subcutaneous, oral, or nasal. The optimal dose for administration and route of administrations are generally determined experimentally.

A method of reducing toxic ß-amyloid levels in the cerebrospinal fluid (CSF) and brain parenchyma of subjects having increased ß-amyloid levels is provided. More specifically, toxic ß-amyloid levels in the cerebrospinal fluid (CSF) and brain parenchyma of subjects is reduced as early as 24 hours of a single parenteral administration of the composition of the present invention.

The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.

Example 1: Construction of the BBB-Fc-L-ABP Fusion Molecules

Fusion molecules comprising:

-   -   a) the FC5 sdAb (SEQ ID NO:14), a murine Fc (SEQ ID NO:48) and         ABP (SEQ ID NO: 30),     -   b) a humanized version of FC5 (FC5-H3; SEQ ID NO: 17), a human         Fc (SEQ ID NO: 49, SEQ ID NO: 50,) and ABP (SEQ ID NO: 30; SEQ         ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID, NO: 34; SEQ ID         NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO:         39; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43;         SEQ ID NO: 44; SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO: 47),     -   c) a humanized version of IGF1R-5 sdAb (IGF1R-5-H2; SEQ ID NO:         26), a murine Fc (SEQ ID NO:48) and ABP (SEQ ID NO: 30)         were prepared. A schematic of the fusion protein construct is         shown in FIGS. 1Ai-1D. The fusion protein comprises a         BBB-crossing single-domain antibody, an Fc fragment and an         amyloid-binding peptide as a dimer.

Example 2: Production of BBB-Fc-L-ABP Fusion Molecules

The constructs FC5-mFc-ABP and humanized FC5(H3)-hFc-ABP described in Example 1 were expressed in CHO cells as dimers, and the expressed compounds were purified on MabSelect Sure affinity columns

Constructs comprising FC5 variants FC5 and FC5-H3 V_(H)H fused to N-terminal of mouse or human Fc antibody fragment fused to ABP variants at their C-terminus as described in example 1 were prepared, expressed, and purified.

The FC5-Fc-ABP variant DNAs (DNA synthesis suppliers) were cloned into mammalian expression vector pTT5 (Durocher 2002). Polyplexes for a final concentration of 1 mg of DNA per liter of cells were pre-formed by mixing a combination of plasmid vector (80%), pTT-AKTdd (15%, activated mutant of Protein Kinase B), and pTTo-GFP (5%, to monitor transfection efficiency) with PEI MAX solution (Polysciences cat no 24765). The PEI:DNA ratio was 4:1 (W:W), both prepared in supplemented F17 medium (4 mM Glutamine, 0.1% Kolliphor). The mixture was incubated for 5 minutes at room temperature prior to addition to the cell culture. The volume of the DNA/PEI polyplexe represent 10% of final culture volume (e.i. 100 ml per 1 L culture). Twenty four hours post-transfection, the cultures were fed with tryptone N1 at a final concentration of 1% (with 40% w/v solution, Organotechnie) and 0.5 mM valproic acid (200 mM solution). The transfection/production were monitored for cell density and viability as well as for productivity titer (mg of Fc per L) and were harvested (supernatant) by centrifugation when the cell viability reach a minimum of 65%. Clarified cell culture medium was filtered through a 0.45 μm membrane prior to its application on a column packed with 5 ml of protein-A MabSelect SuRe resin (GE Healthcare). After loading, the column was washed with 5 volumes of phosphate-buffered saline pH 7.1 (PBS) and the fusion protein was eluted with 100 mM sodium citrate buffer pH 3.0. Fractions containing the eluted fusion protein were pooled and a buffer exchange was performed by loading on a desalting Econo-Pac column (BioRad) equilibrated in PBS. Desalted fusion protein was further purified by Cation Exchange Chromatography (CEX) and sterile-filtered by passing through a Millex GP (Millipore) filter unit (0.22 μm) and aliquoted. CEX fractions were characterized by Mass spectrometric and Western blot analyses as homo- and hetero-dimeric variants.

SDS-PAGE and Aß overlay: Protein samples, prepared in Laemmli sample buffer (heated at 70° C. for non-reducing and at 95° C. for reducing gels, BME or DTT), were separated by SDS-PAGE on 12% Tris-Tricine gels or TGX4-15% gels (BioRad). Gels were either stained with Coomassie blue or the proteins were transferred to PVDF or nitrocellulose membranes for Western blot/Aß overlay assay. The immunoblots were blocked with non-fat dry milk and then exposed to Aß preparations for 45 min at room temperature (50-100 nM) and the bound Aß was detected using 6E10 antibody as described previously (Chakravarthy et al. 2013).

ELISA: Aß-binding assays were carried out as described by Chakravarthy et al (2013). Maxisorp 96-well ELISA plates (Nunc) were coated with (100-500 ng/well) either free ABP (synthetic) or various FC5-ABP constructs overnight at 4° C. in PBS. The wells were blocked with 1% BSA in TBS-T for 30 min and then incubated with Aß₁₋₄₂ preparations consisting predominantly of either monomer and dimers (Mo) or higher oligomers (Oli) in TBS-T at RT for 45 min with gentle agitation. Following three TBS-T washes, bound Aß was detected by incubating HRP-conjugated Aß-specific antibody (6E10 or 4G8) for 90 min at RT in TBS-T. The bound antibody was detected with SureBlue™ TMB reagent kit (KPL) by colorimetric measurement at 450 nm according to manufacturer's instructions.

A Coomassie blue stained gel after separation of FC5 fusion molecules by SDS-PAGE (NR—non-reducing and R—reducing conditions) indicating successful production of recombinant fusion molecule is shown in FIG. 2A. Aß-oligomer binding of free ABP and FC5-Fc-ABP fusion protein by ELISA and Western blot (WB) Overlay assay is shown in FIGS. 2B and 2C. Free or fused ABP was immobilized on ELISA plate by coating samples in phosphate buffered saline (PBS) overnight at 4° C. and exposed to Aß preparations as described by Chakravarthy et al., 2013. Bound Aß was detected with Aß-specific antibody 6E10 or 4G8 (Chakravarthy et al., 2013). The fusion molecules were also separated by SDS-PAGE, transferred to PVDF paper and exposed to Aß oligomers. Bound Aß was detected with specific antibody as above. Results show that ABP retained its Aß oligomer binding ability after fusion with the BBB carrier. Mo: Aß monomers; Oli: Aß Oligomers

Example 3: Binding of BBB-Fc-L-ABP Fusion Molecules to A6 Deposits in AD-TQ Mice (B6.Cq-TQ, Jackson Lab) In Vitro

The constructs produced in Example 2 were submitted to immunohistofluorescence assay to evaluate whether the FC5-Fc-ABP fusion molecules retained the ability to bind naturally-produced amyloid deposits in mouse brain as described (Chakravarthy et al., 2014). Frozen hemi-brains from wild type (Wt) and AD transgenic (AD-Tg) mice were embedded in OCT and 10-μm sections were prepared using a Jung CM 3000 cryostat and stored at −80° C. Tissue sections were thawed and OCT peeled from sections with a razor blade and then incubated with Dako protein blocking reagent for 30 min at room temperature. Blocking agent was removed and sections were gently washed in TBS. IR 800-labelled FC5-mFc-ABP (1:250 dilution of 5.0 μg/μl solution) in antibody diluent was added and incubated for 1 h at room temperature. Sections were then washed twice with TBS, rinsed in Milli Q water, excess rinse solution removed and sections were cover-slipped with Dako Fluorescent Mounting Media. Sections visualized under fluorescence microscope (FIG. 3 ). Selective binding (bright spots) was seen in brain sections from AD-Tg mice that produce Aß deposits and not in brain sections from Wt mice that does not produce amyloid deposits, indicating that ABP in the FC5-Fc-ABP construct retains the ability to bind naturally produced Aß aggregates in AD-Tg mouse brain. In the BBB-Fc-ABP constructs provided, the BBB may be FC5 or an anti-IGF1R antibody.

Example 4: BBB Transmigration of FC5-Fc-L-ABP Fusion Molecules In Vitro

BBB-crossing of FC5-Fc-ABP fusion molecules was assessed in in vitro BBB models from rat and human (FIGS. 4A-4D). Fusion molecules crossing BBB were screened in in vitro BBB permeability assay, using a single-time point for Papp determination. The quantification of variants was done by MRM-ILIS (FIGS. 4A, 4B and 4C).

SV40-immortalized Adult Rat Brain (SV-ARBEC) and Human Brain Endothelial Cells (HBEC) were used to generate an in vitro blood-brain barrier (BBB) model as described (Garberg et al., 2005; Haqqani et al., 2013). Sv-ARBEC (80,000 cells/membrane) were seeded on a 0.1 mg/mL rat tail collagen type I-coated tissue culture inserts (pore size-1 μm; surface area 0.9 cm², Falcon) in 1 ml of growth medium. The bottom chamber of the insert assembly contained 2 ml of growth medium supplemented with the immortalized neonatal rat astrocytes-conditioned medium in a 1:1 (v/v) ratio. Equimolar amounts (5.6 μM) of positive (FC5 constructs) or negative controls (A20.1, a Clostridium difficile toxin A binding V_(H)H; and EG2, an EGFR binding V_(H)H), and Fc-ABP from Example 1 were tested for their ability to cross the rat or human in vitro BBB model. Following exposure of equimolar amounts of the sdAb to the luminal side of the BBB, samples were taken after 15, 30 and 60 min from the abluminal side. The sdAb content of each sample was then quantified by mass spectrometry (multiple reaction monitoring-isotype labeled internal standards; MRM-ILIS) (FIGS. 4A, 4B, 4C).

MRM-ILIS: The methods are all as described in Haqqani et al. (2013). Briefly, to develop the SRM (selected reaction monitoring also known as multiple reaction monitoring (MRM)) assay for V_(H)H, each V_(H)H was first analyzed by nanoLC-MS/MS using data-dependent acquisition to identify all ionizible peptides. For each peptide, the 3 to 5 most intense fragment ions were chosen. An initial SRM assay was developed to monitor these fragments at attomole amounts of the digest (about 100-300 amol). Fragments that showed reproducible intensity ratios at low amounts (i.e., had Pearson r2≥0.95 compared to higher amounts) were considered stable and were chosen for the final SRM assay. To further optimize the assay, elution times for each peptide were also included, with care taken to not choose peptides that have close m/z (mass-to-charge ratio) and elution times.

A typical multiplexed SRM analysis of V_(H)H in cell media or body fluids (serum or cerebrospinal fluid (CSF)) involved spiking known amount of ILIS (0.1-10 nM) followed by injecting 100-400 ng of CSF or cultured media proteins (0.3-1 μL) or about 50-100 ng of serum proteins (1-3 nanoliters) into the nanoLC-MS system. The precursor m/z of each target peptide ion was selected in the ion trap (and the remaining unrelated ions were discarded) at the specified elution time for the target, followed by collision induced dissociation (CID) fragmentation, and selection of only the desired fragment ions in the ion trap for monitoring by the detector. For quantification analysis, raw files generated by the LTQ (ThermoFisher) were converted to the standard mass spectrometry data format mzXML and intensities were extracted using an in-house software called Q-MRM (Quantitative-MRM; see Haqqani et al. 2013), which is a modified version of MatchRx software. For each V_(H)H, extracted-ion chromatograms were generated for each of its fragment ion that consisted of combined intensities within 0.25 Da of the fragment m/z over the entire elution time. To obtain a final intensity value for each fragment, all intensities within 0.5 min of the expected retention times were summed. A V_(H)H was defined as detectable in a sample if the fragments of at least one of its peptides showed the expected intensity ratios, i.e., the final intensity values showed a strong Pearson correlation r≥0.95 and p<0.05 compared with the final intensities values of its corresponding pure V_(H)H.

Samples containing mixtures of V_(H)H (media, serum, CSF) were reduced, alkylated and trypsin-digested as previously described (Haqqani et al., 2012; Gergov et al., 2003). The digests (tryptic peptides) were acidified with acetic acid (5% final concentration) and analyzed on a reversed-phase nanoAcquity UPLC (Waters, Milford, Mass.) coupled to LTQ XL ETD or LTQ Orbitrap ETD mass spectrometer (ThermoFisher, Waltham, Mass.). The desired aliquot of the sample was injected and loaded onto a 300 μm I.D.×0.5 mm 3 μm PepMaps C18 trap (ThermoFisher) then eluted onto a 100 μm I.D.×10 cm 1.7 μm BEH130C18 nanoLC column (Waters) using a gradient from 0%-20% acetonitrile (in 0.1% formic) in 1 minute, 20%-46% in 16 min, and 46%-95% in 1 min at a flow rate of 400 nL/min. The eluted peptides were ionized into the mass spectrometer by electrospray ionization (ESI) for MS/MS and SRM analysis using CID for fragmentation of the peptide ions. The CID was performed with helium as collision gas at normalized collision energy of 35% and 30 ms of activation time. Ion injection times into linear ion trap were adjusted by the instrument using an automatic gain control (AGC) target value of 6×10³ and a maximum accumulation time of 200 ms.

Determination of the apparent permeability coefficient: Quantified values can be directly plotted or the P_(app) (apparent permeability coefficient) values can be determined with the given formula [Qr/dt=cumulative amount in the receiver compartment versus time; A=area of the cell monolayer; C0=initial concentration of the dosing solution] and plotted. The P_(app) value is commonly used to determine the ability of a molecule to cross the BBB. P_(app) values are a measure of the specific permeability of the compound across brain endothelial monolayer.

The specific peptides used for detection and quantification of FC5 constructs are shown in Table 1 below.

TABLE 1 Tryptic MRM Type peptide Protein FC5 ITWGGDNT FC5-mFc-ABP peptide FYSNSVK + ILIS mFc NTEPVLDSDGS FC5-mFc-ABP, peptide YFMYSK host IgGs ABP ASAQASLASK FC5-mFc-ABP peptide IGF1R5-H2 GLEWVATIDWGDGGTR IGF1R5-H2- peptide mFc-ABP IGF1RF-H2 AEDTAVYYCAMAR IGF1R5-H2- peptide mFc-ABP Aβ LVFFAEDVGSNK Host APP, Aβ1-42 or Aβ1-40 Albumin APQVSTPTLVEAAR Host albumin

The samples were also analyzed by Western blot analysis using Fc-specific antibody (FIG. 4D, done in triplicate) as described in Example 2. FC5-mFc-ABP crossed the blood brain barrier as effectively as FC5-mFc, whereas Fc-ABP without the BBB carrier moiety FC5 did not traverse across the brain endothelial cell monolayer. As expected, control single domain antibodies EG2 and A20.1, or control full IgG (anti-HEL) did not cross the blood brain barrier. Similar results were obtained with humanized FC5(H3)-hFc-ABP fusion protein (FIG. 4C and FIG. 4D). Similar results were obtained with IGF1 R5-mFc-ABP ABP (FIGS. 17A and 17B).

Example 5: BBB Transmigration and Pharmacokinetics of FC5-Fc-L-ABP Fusion Molecules In Vivo

The ability of the constructs of Example 2 to transmigrate the blood brain barrier into the brain, specifically into the cerebrospinal fluid (CSF), was evaluated in vivo, as well as to quantify the construct presence in CSF and serum. FC5-mFc-ABP was administered intravenously into rats via tail vein at the indicated doses (2.5, 6.25, 12.5, and 25 mg/kg). Serum and CSF were serially collected. FC5-Fc-ABP levels were quantified using nanoLC-MRM method.

The technique used for multiple sampling of cisterna magna CSF was developed at NRC by modification of previously described methods (Huang et al., 1995; Kornhuber et al., 1986)). All animals were purchased from Charles River Laboratories International, Inc. (Wilmington, Mass., USA). Animals were housed in groups of three in a 12 h light/dark cycle at a temperature of 24° C., a relative humidity of 50±5%, and were allowed free access to food and water. All animal procedures were approved by the NRC's Animal Care Committee and were in compliance with the Canadian Council of Animal Care guidelines. Male Wistar rats aged 8-10 weeks (weight range, 230-250 g) were used in all studies.

In all experiments, test antibodies (FC5 Fc-fusions) were administered intravenously into tail vein in equimolar doses (7 mg/kg). CSF sample collections were made from cisterna magna by needle puncture up to five times over 96 hours. For sample collection rats were briefly and lightly anesthetized with 3% isoflurane, placed in a stereotaxic frame with the head rotated downward at a 45° angle. A 2-cm midline incision between the ears beginning at the occipital crest was made and muscles separated to expose dura mater covering cisternae magna. A 27G butterfly needle (QiuckMedical, Cat #SV27EL) with tubing attached to 1 ml syringe was used to puncture dura and aspirate the ˜20 I of CSF. The CSF was then transferred into the sample glass vial (Waters, Cat #186000384c) and placed in −80° C. freezer until further analysis.

Blood samples were collected from the tail vein in a commercially available tube (BD microtainer, Cat #365956). After clotting at room temperature for 15-30 minutes, the clot was removed by centrifuging at 1100 rcf (3422 rpm) for 10 min; serum was then transferred into a clean glass vial (Waters, Cat #186000384c), frozen on dry ice and stored at −80° C. until further analysis. At the end of collection, rats were sacrificed by cardiac puncture. Blood and CSF PK analyses were performed using WinLin 6.0 program.

Serum and CSF samples were analyzed by mass spectrometry and nanoLC-SRM based quantification as described in Example 4 using peptide signatures shown in Table 1.

CSF collection is a delicate procedure during which CSF can be easily contaminated with blood. Since the amounts of V_(H)H s were expected to be much smaller in the CSF (<0.1%) than blood, even a slight contamination with blood could seriously compromise the value of an individual CSF sample. It was therefore necessary to develop stringent exclusion criteria for blood-contaminated CSF samples. To evaluate blood-CSF albumin ratio, a nanoLC-SRM method was developed for quantifying albumin levels in plasma and CSF. An albumin peptide APQVSTPTLVEAAR was selected based on its unique retention time and m/z value (Mol Pharm) in order to have minimum interference with other peptide peaks in the multiplex assay. The intensity of the peptide was quantified in both CSF and plasma samples using SRM as described above. The albumin ratio was calculated as follows for each rat:

Albumin Ratio=Intensity per nL of plasma analyzed/Intensity per nL of CSF analyzed

A ratio of 1500 and below was considered as blood contaminated.

As shown in FIG. 5 , FC5-mFc-ABP appeared in the CSF in a time- and dose-dependent manner with Cmax between 12 and 24 h, indicating transport of ABP by FC5 into brain and CSF compartments in vivo (A). Serum PK parameters (FIG. 5 and Table 2 below) show that alpha- and beta-half-life of FC5-mFc-ABP is similar to that of a full IgG (a benchmark antibody containing rat Fc) and is substantially higher than that of ABP or FC5 or FC5-ABP without Fc.

TABLE 2 Dose range: 2.5 mg/kg. 6 mg/kg, 12 mg/kg, 25 mg/kg, mAb Parameter Estimate Units CV (%) V1 114.6 mL/kg 2.3 V2 74.4 mL/kg 6.5 CL 0.664 mL/(kg*hr) 1.9 CLd 2.682 mL/(kg*hr) 41.8 Alpha T_(1/2) 11.2055 hr 38.0 Beta T_(1/2) 205.279 hr 2.2 Vss 189.0 mL/kg 1.4 FC5mFc-ABP (FC5 domain} Parameter Estimate Units CV (%) V1 144.5 mL/kg 2.2 V2 218.4 mL/kg 27.2 CL 1.056 mL/(kg*hr) 26.5 CLd 3.789 mL/(kg*hr) 10.6 Alpha T_(1/2) 14.36 hr 14.3 Beta T_(1/2) 263.8 hr 41.3 Vss 362.9 mL/kg 16.3 Serum Half Life (Both Distribution Phase and Terminal) are Similar Between ‘Benchmark mAb’ and FC5mFc-car90

Example 6: Delivery of FC5-ABP Construct to the Brain in Non-Rodent Larger Animal

Serum and CSF PK profile of FC5-mFc-ABP was assessed in beagle dog. FC5-mFc-ABP was administered by intravenous injection to 10-12-year old beagle dogs and serum and CSF were serially collected and analyzed by nanoLC-MRM (left panel) and by Western blot using Fc-specific antibody (FIG. 6B) as described above in Example 5. Asterisks indicates blood-contaminated sample (not shown in MRM analyses). As can be seen, FC5-mFc-ABP appeared in the CSF in a time-dependent manner indicating transport of ABP by FC5 across dog blood brain barrier in vivo, confirming the translational nature of the BBB carrier. The PK parameters and CSF exposure were analyzed by WinNonlin software and are shown in Table 3 below.

TABLE 3 Dog 1 Dog 2 Mean Mean Parameter Unit Estimate SD Estimate SD t1/2 h 96 21 89 21 AUC 0-t ng/ml * h 8.80E+06 1.99E+06 8.84E+06 2.16E+06 AUC 0-inf_obs ng/ml * h 1.01E+07 2.21E+06 1.04E+07 2.76E+06 Cl_obs ml/h/kg 1.54 0.36 1.53 0.48 Vss_obs ml/h/kg 193 7 205 10 Alpha half life h 19 10 13 8.5 Beta half life h 114 37 166 151 AUC_(0-t,cst)/ % 0.88 0.09 0.52 0.04 AUC_(0-t, serum)

Example 7: BBB Permeability

BBB permeability and CSF appearance of FC5 fused with human Fc (hFc) and chemically linked to ABP (FC5-hFc-ABP) in vivo (rat model). FC5-hFc was linked with ABP-cystamide using a heterobifunctional cross-linker sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) according manufacturer's instructions (ThermoFisher Scientific). Chemically conjugated molecule was administered intravenously into rats via tail vein at 6.25 mg/kg and serum and CSF samples were collected at 4 and 24 hrs and analyzed as described in FIG. 5 . FC5-hFc-ABP appears in the CSF in a time-dependent manner, however Fc-ABP without the BBB carrier does not, confirming FC5-mediated transport of ABP across the blood brain barrier, and as illustrated in FIG. 7 .

Example 8: Delivery of BBB-Fc-L-ABP Construct to the Brain

The ability of the FC5-Fc-ABP constructs of Example 2 to transmigrate the blood brain barrier and penetrate the brain parenchyma in vivo was assessed in mice.

FC5-mFc-L-ABP was administered by intravenous injection via tail vein at either 15 mg/kg to wild type (WT) and AD-transgenic (AD-Tg, B6.Cg-Tg, Jackson Lab) mice (FIGS. 8A and 8B) or at 7.5, 15 and 30 mg/kg to AD-Tg mice, and circulated for 4 and 24h. Mice were then thoroughly perfused with 10 ml of heparinized (100U/ml) saline at a rate of 1 ml/min via the left common carotid artery to facilitate specific perfusion of the brain. Brains were then removed, and hippocampal and cortical tissues were dissected and immediately frozen and stored at −80° C. until use. Frozen tissue was homogenized in ice-cold homogenization buffer containing 50 mM Tris-HCl pH 8, 150 mM NaCl and protease inhibitor cocktail (Sigma-Aldrich, Oakville, ON) using Dounce homogenizer (10-12 stroke at 4° C.). Samples were then sonicated three times for 10 s each at 4° C. and insoluble material was removed (10,000×g for 10 min at 4° C.). The supernatant was analyzed for protein content, and about 0.5 μg of protein was used for SRM analysis (FIG. 8A) using methods described in Example 4 and peptide signatures shown in Table 1. Samples were also analysed by Western blot using mFc-specific antibodies (FIGS. 8B and 8C). Specific “signature” peptides belonging to all the three components of the fusion molecule (FC5, Fc and ABP) were detected by MRM in both cortex and hippocampus, indicating that FC5 carrier successfully delivered ABP to the target areas of the brain (only data from FC5 peptide is shown in FIG. 8A). Measured levels ranged between 750-1400 ng/g brain tissue at different time points, compared to ˜50 ng/g tissue typically measured for control single-domain antibody A20.1 fused to Fc, or Fc fragment alone. This was further confirmed by Western blot analysis probing for Fc and ABP in tissue extracts (FIGS. 8B and 8C). No protein signal of the fusion molecule was detected in animals receiving just saline by Western blot. There was a dose-dependent increase in FC5-mFc-ABP levels detected by Western blot in the target regions of the brain (FIG. 8C). These results clearly indicate that FC5 successfully delivers ABP to the target regions of the brain (i.e., the hippocampus and cortex) in wild type (WT) and AD-Tg mice.

Example 9: Clearance of A3 from Mouse Brain

To evaluate the efficacy of ABP on the amyloid burden in Tg mice, the results of treatment with ABP alone or FC5-ABP construct were compared.

Comparison between treatment with ABP alone (FIG. 9A) or ABP fused with BBB carrier FC5 (FIGS. 9B and 9C). Two different AD Tg mouse models, triple transgenic (3×Tg-AD, sv129/C57BL6 mice harboring PS1M146V, APP_(Swe) and tauP301L transgenes, Dr. F. M. LaFerla, University of California) and double transgenic (B6.Cg-Tg, harboring PSEN1dE9 and APP_(Swe) transgenes, Jackson Lab) were used; mice were dosed subcutaneously (sc) with 300 nmol/kg of free ABP every second day over a 3-month or a 2-month period, respectively. At the end of the treatment period, Aß levels in the brain were measured by ELISA using a commercial assay kit (InVitrogen, KHB3544) according to manufacturer's assay procedure. The treatment with ABP alone resulted in 25-50% reduction in brain Aß after 2-3 months of multiple treatments (every second day) (9A). The FC5-mFc-ABP construct was injected intravenously into double-transgenic AD mice (B6.Cg-Tg, 15 mg/kg; equivalent of 220 nmol/kg) and brain Aß levels were measured by both ELISA and nanoLC-MRM 24 h after injection as described above in Example 8. Unexpectedly, about 50% amyloid reduction was observed within 24 hr of treatment with FC5-mFc-ABP (FIG. 9B), indicating that efficient brain delivery of ABP by FC5 dramatically increased the efficacy of ABP in reducing brain Aß levels. CSF analysis also indicated a significant decrease in Aß₁₋₄₂ levels within 24 hrs following FC5-mFc-ABP treatment (FIG. 9C). The Aß peptide sequence detected by MRM (SEQ: LVFFAEDVGSNK, Table 1/ELISA analyses is remote/different from the Aβ epitope recognized by ABP (therefore, not interfering with its quantification by either ELISA or MRM).

Example 10: Introduction of Fc Component into FC5-ABP Construct Enhances its Serum Half-Life

FC5-ABP (FC5 SEQ ID NO 17; and ABP SEQ ID NO 36) and FC5-hFc-ABP (FC5 SEQ ID NO 17; hFc 1x7 SEQ ID NO 49 and ABP SEQ ID NO 36] constructs were produced in CHO cells as described in Example 2. Serum PK was determined as described in Example 5. FC5-ABP and FC5-Fc-ABP constructs were administered intravenously into rat tail vein at 15 mg/kg. Serum was serially collected and FC5-ABP and FC5-Fc-ABP levels were quantified by direct ELISA using FC5- and ABP-specific antibodies. Serum samples were diluted (1:5,000) in phosphate-buffered saline (PBS) and applied to Maxisorb plates and incubated overnight at 4° C. ELISA plates were washed 3×100 μl PBS and blocked with 1% BSA in TBST for 30 min at room temperature (RT). Blocking solution was removed and the plates were incubated with HRP-conjugated FC5 monoclonal antibody (90 min). Following incubation and wash, 100 μl SureBlue reagent was added and incubated in the dark at RT for 10-15 min. At the end of reaction, 100 μl 1 M HCl was added and the developed colour was read at 450 nm in a plate reader. As shown in FIGS. 10A-10B, FC5-ABP without Fc was very rapidly cleared in the serum (within an hr) compared to FC5-ABP construct containing Fc component (FC5-Fc-ABP). This assay was also repeated with ABP-specific antibody with similar results. After sample application, ELISA plates were incubated first with ABP rabbit polyclonal antibody (90 min), followed by HRP-conjugated rabbit secondary antibody (30 min) and the bound antibody was detected as described above (data not shown).

Example 11: Clearance of Aß from Rat Brain

AD-Tg rats were dosed with either saline or FC5-mFc-ABP via tail vein every week over a period of four weeks (loading dose of 30 mg/kg and subsequent four weekly doses of 15 mg/kg). CSF levels of FC5-mFc-ABP and Aß were analyzed by nanoLC MRM. Before and after four weeks of treatment, brain Aß levels were determined by PET scan using a specific Aß-binding agent [18F] NAV4694. FC5-mFc-ABP reduced CSF Aß level in rats within 24 hrs. An inverse relationship between the CSF levels of FC5-mFc-ABP and Aß was observed, as in Tg mice, suggesting target engagement and rapid clearance of Aß by ABP delivered to the brain and CSF by FC5 (FIG. 11A and FIG. 11B). This was further corroborated by PET scan which clearly indicated a significant reduction (30-50%) of rat brain Aß levels following four weeks of treatment with FC5-mFc-ABP (FIG. 11C).

Example 12: Increased Hippocampal Volume and Improved Neuronal Connectivity

In the experiment described in Example 11, saline- and FC5-mFc-ABP-treated Tg mice were subjected volumetric and functional Magnetic Resonance Imaging (MRI) before after the treatment. Volumetric MRI (FIG. 12A) showed an increased hippocampal volume in ABP-treated Tg mice compared to saline-treated controls suggesting that ABP treatment arrested hippocampal atrophy. Functional MRI (FIG. 12B) showed improved connectivity in anterior cingulated cortex in ABP-treated Tg mice compared to saline-treated controls suggesting restoration of neuronal connectivity. This data confirms the significance, efficacy and superior therapeutic advantages now provided.

Example 13: FC5-mFc2a-ABP Treatment Shows Decreased Levels of CSF Aβ in Dogs

As described in Example 6, serum and CSF PK profile of FC5-mFc-ABP was assessed in beagle dog with two dose (15 mg/kg and 30 mg/kg. In addition to measuring serum and CSF levels of FC5-mFc2a-ABP by nanoLC-MRM, CSF levels of Aβ was also measured by nanoLC-MRM as described in Example 9. As can be seen, FC5-mFc-ABP appeared in the CSF in a dose- and time-dependent manner (FIG. 13B). Most importantly, as seen Tg mice and Tg rat, there was a significant decrease in CSF Aβ level that was inversely proportional to CSF FC5-mFc2a-ABP levels.

Example 14: Generation of ABP Fusion Molecule with a Different BBB Carrier

To assess the versatility of ABP fusion molecule, ABP was successfully fused with another humanized BBB carrier IGF1 R5 (H2). As shown in FIG. 12 , the bi-functionality of the molecule was retained, ABP's ability to bind Aβ oligomer (ELISA and overlay assays) and also IGF1 R5's ability deliver ABP across BBB model in vitro (data not shown). This clearly indicates that ABP can be fused to different BBB-crossing single-domain antibodies to be delivered to the brain.

Example 15: Aβ Oligomer Binding by Different BBB-Crossing Single-Domain Antibody-Fc-ABP Constructs

Various FC5-Fc-ABP constructs with modified ABPs (site-specific mutations or removal of C-terminus portion of the molecule as indicated by SEQ ID Nos shown in Table 1. and FIG. 15A-15C) are provided. As shown in FIGS. 15A-15C, all constructs retained similar potency in binding Aβ oligomers by ELISA method.

Example 16: Production of FC5-hFc1X7-L-ABP with Specific Mutations to Improve Stability and Bio-Manufacturability

FC5-hFc1X7-L-ABP carrying specific mutations (ABP, SEQ ID NO 35; ABP, SEQ ID NO 36) were produced in CHO cells and separated on SDS-PGE under reducing (R) and non-reducing (NR) conditions and stained with Coomassie, blue as described in FIG. 2A-2C. Separated protein transferred to nitrocellulose membrane and immunoblotted with FC5-specific, hFc-specific and ABP-specific antibodies. In another set, Aß-binding of ABP in the fusion molecule was also tested by overlay assay. Bound Aß was detected with Aß-specific antibody 6E10. As can be seen, systematic modification of ABP with specific mutations (as shown here, for example, ABP SEQ ID NO: 35 and, ABP SEQ ID NO: 36) substantially enhanced the stability of the molecule generated, as is clearly indicated herein, for example, by a single protein band under reducing and non-reducing conditions (compare with other ABP constructs of FIG. 2A, wherein double protein bands can be seen). This substantial enhancement in the stability of the fusion molecule advantageously facilitates the bio-manufacturability of a homogeneous molecule.

Example 17: BBB Permeability of Various FC5-Fc-L-ABP Constructs and IGF1R5-Fc-ABP Construct In Vitro

BBB-crossing was assessed in in vitro rat BBB model as described in FIGS. 4A-4D and molecules crossing the blood brain barrier were detected by nanoLC-MRM method. All ABP variants fused to humanized FC5 and IGF1R carriers crossed the BBB effectively. As expected, A20.1, a non-BBB permeable sdAb did not cross BBB, and likewise, ABP fused to A20.1 did not permeate the BBB (FIG. 17A). In FIG. 17B, it is shown that “finger-print” peptides for all the three components of the fusion molecule, FC5, Fc and ABP were detected by nanoLC-MRM.

The embodiments and examples described herein are illustrative and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Furthermore, the discussed combination of features might not be necessary for the inventive solution.

Example 18

Humanized FC5-Fc-ABP construct [FC5(H3)-hFc-ABP (with ABP SEQ ID NO: 35 and ABP SEQ ID NO: 36)] crosses in vitro rat blood brain barrier intact. Blood brain barrier-crossing of humanized FC5-ABP fusion molecules were assessed as described in Example 4. Fusion molecules crossing the blood brain barrier were analyzed by Western blot and ELISA methods. Immunoblots were probed with hFc-specific-, and ABP-specific antibodies. Both the antibodies recognized the molecule crossing the BBB (bottom chamber) and the molecular size was identical to that of FC5-ABP fusion construct that was applied to in vitro BBB (top chamber) indicating that the molecule that crossed the BBB remained intact. This was substantiated by sandwich ELISA assay where in the BBB-crossed molecule was captured with FC5-specific antibody and the captured molecule was detected with ABP antibody.

Example 19

Humanized FC5-Fc-ABP construct [FC5(H3)-hFc-L-ABP (with ABP SEQ ID NO: 35 and ABP SEQ ID NO: 36)] is transported across the BBB in vivo and delivered to the brain intact by FC5. Blood brain barrier-crossing and brain delivery of humanized FC5-ABP fusion molecule in mice was assessed as described in Example 8. Four hrs following intravenous administration of the molecule and intracardiac perfusion, brain was removed and the cortices were extracted in RIPA buffer and the presence of injected FC5-ABP fusion molecule was detected by Western blot probed with ABP-specific antibody and by sandwich ELISA by capturing the molecule with FC5-specific antibody and detecting with ABP-specific antibody. Western blot revealed the presence of full-length FC5-ABP construct in the cortex. This was substantiated by sandwich ELISA which revealed the intact nature of the molecule as indicated by the ability to capture the molecule by FC5-specific antibody and detect the captured molecule with ABP-specific antibody.

Example 20: Ex-Vivo (A) and In Vivo (B) Binding of Humanized FC5-Fc-ABP Construct [FC5(H3)-hFc-ABP (ABP SEQ ID NO 36] (Target Engagement)

Immunohistochemistry was carried out as described in Example 3. Brain sections from AD-transgenic mice were incubated with FC5(H3)-hFc1X7-ABP (ABP, SEQ ID NO: 36) and the bound fusion molecule was visualized with HRP-conjugated FC5-specific antibody. As a negative control, sections were incubated with either no construct, or FC5-hFc construct without fused ABP. Briefly, Formalin-fixed 40 μm free-floating sections containing cortex and hippocampus from APP/PS1 transgenic mouse were subjected to antigen retrieval in 10 mM sodium citrate buffer (pH 9) at 80° C. for 30 minutes then cooled to room temperature. Sections were rinsed in PBS, treated with 3% H₂O₂ in PBS for 30 minutes to block endogenous peroxidase, then rinsed again. Following a one hour incubation in Dako serum free protein block containing 0.3% triton X-100, sections were incubated with FC5(H3)-hFc1x7-ABP or the molar equivalent of FC5-hFc1x7 in Dako diluent containing 0.3% triton X-100 for 90 minutes at room temperature. After thoroughly rinsing in PBS, the sections were incubated with anti-FC5-HRP in Dako diluent for 60 minutes at room temperature, rinsed again, and then developed using Vector Immpact DAB following the kit directions. The sections were placed on Superfrost plus slides, allowed to air dry overnight, then rehydrated, counterstained with methyl green, dipped in acetone/0.05% acetic acid (v/v) and dehydrated, cleared and coverslipped with Permount. Selective binding (black spots) was seen with FC5(H3)-hFc1X7-ABP construct but not with FC5(H3)-hFc1X7 without ABP, indicating ABP-dependent binding of Aβ deposits (target engagement) in the brain. No binding was seen in brain sections from wild type mice that does not produce amyloid deposits (data not shown).

Similar binding of Aß deposits were detected following intra-hippocampal injection of FC5(H3)-hFc-ABP construct into AD transgenic mice (B). Four hours after intra-hippocampal injection, Tg mice were perfused and the brains were removed and sectioned. FC5(H3)-hFc-ABP binding to Aß deposits (shiny white dots) were visualised with ABP-specific polyclonal antibody. Brain sections were first incubated with ABP-specific monoclonal antibody followed by Alexa-647 conjugated anti rabbit-Fc secondary antibody

Example 21

Target engagement by humanized FC5-Fc-ABP construct (FC5(H3)-hFc-ABP (SEQ ID NO: 54) in vivo. Fluorophore-labelled (FIG. 18A) or naïve FC5-ABP (FIG. 18B) fusion molecule was microinjected into the hippocampal region of wild-type (Wt) and AD-Tg (Tg) mice. 30 min after microinjection of fluorophore-labelled FC5-ABP fusion molecule, brains were removed, sectioned and observed under fluorescence microscope. As shown in 21A, injected molecule bound to Aß deposits as confirmed by its co-localization with Aß-specific antibody. In a parallel study, four hrs after intra-hippocampal injection of naïve FC5-ABP fusion molecule, hippocampal formation from injected (ips) and non-injected (con) regions were collected and homogenized in Tris-buffered saline. Hippocampal extracts were subjected to sandwich ELISA with FC5 antibody as capturing antibody and either ABP or Aß-specific antibody as detection antibody. As shown in 21B, microinjected FC5-ABP molecule remained intact and ABP was able to engage and bind the target Aβ in vivo as indicated by the presence of Aβ in the pulled-down complex detected by Aß-specific antibody.

Example 22: PK/PD Comparison Between Non-Humanized and Humanized FC5-Fc-L-ABP Constructs

FC5-mFc2a-ABP or FC5(H3)-hFc1x7-ABP was administered intravenously into rats via tail vein injection at 15 mg/kg as described for FIG. 5 . Serum and CSF were serially collected. FC5-Fc-ABP levels were quantified using nanoLC-MRM method. As shown in FIG. 22A, serum and CSF PK profile were very similar for non-humanized and humanized constructs. FC5-mFc2a-ABP or FC5(H3)-hFc1x7-ABP was administered intravenously into Tg mice via tail vein injection at 15 mg/kg as described for FIG. 9B. FC5-Fc-ABP and Aß levels in the CSF were measured by nanoLC-MRM as described in FIG. 9B. As shown in FIG. 22B, the levels of non-humanized and humanized FC5-Fc-ABP in the CSF were similar, and most importantly, changes (decrease) in CSF Aß levels were also very similar, indicating that humanization of FC5-Fc-ABP construct did not affect the PK and PD profile of the fusion construct.

Example 23: Generation of C-Terminally Cleaved Products During the Production of FC5-(H3)-hFc1x7-ABP (6G) Fusion Protein (SEQ ID NO: 56) in CHO Cells

The fusion protein was produced in stable CHO cell line as described in Examples 2 and 16. Following purification using Protein A affinity column (Examples 2), the fusion protein was further purified using Cation Exchange (CEX) Chromatography. CEX Chromatography profile revealed the presence of variants of FC5-(H3)-hFc1x7-ABP (6G). Upon Western blot analyses with anti-hFc antibody (FIG. 23 A) and Mass spectroscopy (23B) of each CEX fraction, it was determined that the generation of variants of the fusion protein was due to the C-terminal cleavage of the ABP peptide during the production. The C-terminal cleavage occurs at specific sites of the ABP sequence in FC5-(H3)-hFc1x7-ABP (6G) protein, as illustrated in SEQ ID NO: 37 to SEQ ID NO: 43, which are C-terminally cleaved peptides of the SEQ ID NO: 36 ABP sequence. The C-terminal cleavage products of SEQ ID NO: 36 or an ABP having SEQ ID NO: 47 showed unexpected stability and β-amyloid binding activity when incorporated in a fusion protein in a dimeric format. Moreover, such C-terminal cleavage products advantageously maintained the equivalent biological function of ß-amyloid binding activity (EC50, FIG. 23 A) as assessed by ELISA and Western blot Aß overlay assays (as described in Example 2), as compared to the functional homodimeric forms. These C-terminally cleaved products advantageously increase the manufacturability and isolatable yield of functional and stable β-amyloid binding products. These stable cleavage products are not obvious and are advantageous with respect to β-amyloid binding functional peptides and corresponding fusion peptides and dimer yields. Thus, C-terminal cleavage of ABP during production in CHO-cells resulted in the generation of bifunctional homodimers, bifunctional heterodimers (both the ABP arms are functional and may contain any ABP and/or any C-terminally cleaved but functional ABP) and monofunctional heterodimers (one functional ABP in the dimer) of the fusion proteins as depicted in FIGS. 1Ai, 1Aii and 1Aiii. The precise nature of the cleavage products of the fusion protein (SEQ ID NO: 56) was not obvious until detailed analyses were carried out and could not have been predicted in biomanufacturing and protein production. Nor was it known or obvious that the now-defined C-terminal cleavage products would advantageously bind β-amyloid and be stable peptides. The ß-amyloid binding activity of the CEX fractions containing a mixture of bifunctional homodimers, and monofunctional and bifunctional heterodimers (PEAK 2, FIG. 23A, (EC 50 19 nM) was very similar to that of the bifunctional homodimer (PEAK 3, FIG. 23A, EC 50 13 nM), demonstrating that the heterodimer and homodimer fractions (PEAKS 2 and 3 of the CEX) can be combined without significantly losing ß-amyloid binding activity. This substantially and advantageously increases the yield and isolatability of FC5(H3)-hFc1x7-ABP as a fully functional, stable fusion protein during large-scale biomanufacturing. This has been demonstrated in FIG. 23C, which shows that modified CEX conditions generate a fraction with high yield, PEAK 2, FIG. 23C, representing 86.4% of the total protein applied to the column. A typical mass spectrometric analysis of PEAK 2 consists of a mixture of the homodimer of SEQ ID NO: 56, (MW 87,695) and heterodimers comprising SEQ ID NO:56 and SEQ ID NO: 57 (MW=87,470); SEQ ID NO: 56 and SEQ ID NO: 60, (MW=87,085) and SEQ ID NO: 56 and a fully cleaved, non-functional ABP, (MW=84,040) as shown in FIG. 23B. The ß-amyloid binding activity (EC 50) of this combined pool (fractions B3 to B6) was very similar to other CEX fractions containing either predominantly homodimers (C10-D2, E12) or heterodimer forms (C6-C8) as shown in FIGS. 23A-23C.

Example 24: Brain Delivery of the FC5(H3)-hFc1x7-ABP Fusion Protein Containing a Mixture of Homo- and Hetero-Dimers

The transport of FC5(H3)-hFc1x7-ABP across the blood-brain barrier and delivery to the target regions of the brain was assessed as described in Examples 8 and 19. FC5(H3)-hFc1x7-ABP fusion protein (as a mixture of homo and heterodimers from Example 23, was administered via tail vein of wild-type (wt) and Tg mice at 15 mg/kg. After 4 hrs (data not shown) or 24 hrs following the injection, cortical and hippocampal regions of the brain were dissected and assessed as described in Example 8 and 19. As shown in FIGS. 24A-24B, FC5(H3)-hFc1x7-ABP fusion protein was detected in the target regions as assessed by sandwich ELISA (FIG. 24 A) and western blot analysis (FIG. 24 B). The results are very similar to the brain delivery of predominantly homodimeric form of FC5(H3)-hFc1x7-ABP (SEQ ID NO: 56, see FIGS. 19 1.A-1.B and 19 2.A-2.B), further confirming that the heterodimeric form of FC5(H3)-hFc1x7-ABP in the mixture does not affect blood-brain crossing and brain delivery of the fusion protein.

Example 25: CSF Exposure of the FC5(H3)-hFc1x7-ABP Fusion Protein Containing a Mixture of Homo- and Hetero-Dimers and its Effect on CSF 3-Amyloid

24 hrs following intra-venous administration of injection of FC5(H3)-hFc1x7-ABP into Tg mice CSF was collected and the levels of FC5(H3)-hFc1x7-ABP and ß-amyloid were measured by MRM as described in Examples 5 and 22. As shown in FIG. 25 , CSF exposure of FC5(H3)-hFc1x7-ABP and changes (decrease) in CSF Aß levels are very similar to that seen with predominantly homodimeric form of FC5(H3)-hFc1x7-ABP (SEQ ID NO: 56, see FIGS. 22A-22B), further confirming FC5(H3)-hFc1x7-ABP in the mixture does not affect function efficacy of FC5(H3)-hFc1x7-ABP fusion protein.

SEQUENCES SEQ ID NO: Sequence Description 1 GFKITHYTMG CDR1 FC5 2 RITWGGDNTFYSNSVKG CDR2 FC5 3 GSTSTATPLRVDY CDR3 FC5 4 EYPSNFYA CDR1 IGF1R-3 5 VSRDGLTT CDR2 IGF1R-3 6 AIVITGVWNKVDVNSRSYHY CDR3 IGF1R-3 7 GGTVSPTA CDR1 IGF1R-4 8 ITWSRGTT CDR2 IGF1R-4 9 AASTFLRILPEESAYTY CDR3 IGF1R-4 10 GRTIDNYA CDR1 IGF1R-5 11 IDWGDGGX; where X is A or T CDR2 IGF1R-5 12 AMARQSRVNLDVARYDY CDR3 IGF1R-5 13 X₁VQLVX₂SGGGLVQPGGSLRLSCAASGFKITHYTMGWX₃RQAPGKX₄ Humanized FC5 X₅EX₆VSRITWGGDNTFYSNSVKGRFTISRDNSKNTX₇YLQMNSLRAE consensus DTAVYYCAAGSTSTATPLRVDYWGQGTLVTVSS, where X₁ = D or E, X₂ = A or E, X₃ = F or V, X₄ = E or G, X₅ = R or L, X₆ = F or W, X₇ = L or V; 14 DVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKERE FC5 FVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTA DYYCAAGSTSTATPLRVDYWGKGTQVTVSS 15 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLE FC5-H1 WVSRITWGGDNTFYSNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCAAGSTSTATPLRVDYWGQGTLVTVSS 16 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWVRQAPGKGLE FC5-H2 WVSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAV YYCAAGSTSTATPLRVDYWGQGTLVTVSS 17 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3 VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAV YYCAAGSTSTATPLRVDYWGQGTLVTVSS 18 X₁VX₂LX₃ESGGGLVQX₄GGSLRLSCX₅ASEYPSNFYAMSWX₆RQAPGK IGF1R-3 X₇X₈EX₉VX₁₀GVSRDGLTTLYADSVKGRFTX₁₁SRDNX₁₂KNTX₁₃X₁₄LQM Consensus NSX₁₅X₁₆AEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTX₁₇VTVSS where X₁ is E or Q; X₂ is K or Q; X₃ is V or E; X₄ is A or P; X₅ is V or A; X₆ is For V; X₇ is E or G; X₈ is R or L; X₉ is F or W; X₁₀ is A or S; X₁₁ is M or 1; X₁₂ is A or S; X₁₃ is V or L; X₁₄ is D or Y; X₁₅ is V or L; X₁₆ is K or R; and X₁₇ is Q or L, 19 QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMSWFRQAPGKERE IGF1R-3 FVAGVSRDGLTTLYADSVKGRFTMSRDNAKNTVDLQMNSVKAEDTAV YYCAIVITGVWNKVDVNSRSYHYWGQGTQVTVSS 20 EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKERE IGF1R-3-H5 FVSGVSRDGLTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS 21 X₁VX₂LX₃ESGGGLVQX₄GGSLRLSCX₅X₆SGGTVSPTAMGWX₇RQAPGKX₈ IGF1R-4 X₉EX₁₀VX₁₁HITWSRGTTRX₁₂ASSVKX₁₃RFTISRDX₁₄X₁₅KNTX₁₆YLQ Consensus MNSLX₁₇X₁₈EDTAVYYCAASTFLRILPEESAYTYWGQGT X₁₉VTVSS, where X₁ is E or Q; X₂ is K or Q; X₃ is V or E; X₄ is A or P; X₅ is A or E; X₆ is V or A; X₇ is V or F; X₈ is G or E; X₉ is L or R; X₁₀ is For W; X₁₁ is G or S; X₁₂ is V or Y; X₁₃ is D or G; X₁₄ is N or S; X₁₅ is A or S; X₁₆ is L or V; X₁₇ is K or R; X₁₈ is A or S; and X₁₉ is L or Q, 22 QVKLEESGGGLVQAGGSLRLSCEVSGGTVSPTAMGWFRQAPGKERE IGF1R-4 FVGHITWSRGTTRVASSVKDRFTISRDSAKNTVYLQMNSLKSEDTAVY YCAASTFLRILPEESAYTYWGQGTQVTVSS 23 QVQLVESGGGLVQPGGSLRLSCAVSGGTVSPTAMGWFRQAPGKGLE IGF1R-4-H3 FVGHITWSRGTTRYASSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVY YCAASTFLRILPEESAYTYWGQGTLVTVSS 24 X₁VX₂LX₃ESGGGLVQX₄GGSLRLSCAASGRTIDNYAMAWX₅RQAPGKX₆ IGF1R-5 X₇EX₈VX₉TIDWGDGGX₁₀RYANSVKGRFTISRDNX₁₁KX₁₂TX₁₃YLQMNX₁₄ Consensus LX₁₅X₁₆EDTAVYX₁₇CAMARQSRVNLDVARYDYWGQGTX₁₈VTVSS, where X₁ is E or Q; X₂ is K or Q; X₃ is V or E; X₄ is A or P; X₅ is V or S; X₆ is D or G; X₇ is L or R; X₈ is F or W; X₉ is A or S; X₁₀ is A or T; X₁₁ is A or S; X₁₂ is G or N; X₁₃ is M or L; X₁₄ is N or R; X₁₅ is E or R; X₁₆ is P or A; X₁₇ is S or Y; and X₁₈ is Q or L, 25 QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPG IGF1R-5 KDREFVATIDWGDGGARYANSVKGRFTISRDNAKGTMYLQMN NLEPEDTAVYSCAMARQSRVNLDVARYDYWGQGTQVTVSS 26 QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPG IGF1R-5-H2 KGLEWVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMN SLRAEDTAVYYCAMARQSRVNLDVARYDYWGQGTLVTVSS 27 SGKTEYMAFPKPFESSSSIGAEKPRNKKLPEEEVESSRTPWLYEQEG PK-4 (174 aa) EVEKPFIKTGFSVSVEKSTSSNRKNQLDTNGRRRQFDEESLESFSSMP DPVDPTTVTKTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKN ITHARRILQQSNRNACNEAPETGSDFSMFEA 28 FSSMPDPVDPTTVTKTFKTRKASAQASLASKDKTPKSKSK P4 peptide (40 aa) 29 KDKTPKSKSKKRNSTQLKSRVKNITHARRILQQSNRNACN P5 peptide (40 aa) 30 KTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVKNI P4-5 peptide (ABP, 40 aa) 31 X₁TFX₂TX₃X₄ASAQASLASKDKTPKSKSKKX₅X₆STQLX₇S ABP consensus X₈VX₉NX₁₀ (39 or 40 aa) here X₁ = K, G or A, X₁ = G or A, X₂ = K, G or V, X₃ = R, G or A, X₄ = K, G or A, X₅ = R, G or V, X₆ = N, G or V, X₇ = K, G or V, X₈ = R, G or A, X₉ = K, G or A, X₁₀ = I or no amino acid (ie an —OH at the C-terminus of a 39 aa peptide) (SEQ ID NO: 31) 32 KTFKTRKASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI [ABP(G)](40 aa) 33 KTFKTRKASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI [ABP(GG)](40 aa) 34 KTFKTRGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI [ABP(RG-G)] (40 aa) 35 KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI [ABP (GG-G)] (40 aa) 36 GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI [ABP(6G)](40 aa) 37 GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVK [ABP(6G)]c1 (38 aa) 38 GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV [ABP(6G)]c2 (37 aa) 39 GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSR [ABP(6G)]c3 (36 aa) 40 GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS [ABP (6G)] c4 (35 aa) 41 GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLK [ABP(6G)]c5 (34 aa) 42 GTFGTGGASAQASLASKDKTPKSKSKKGGSTQL [ABP(6G)]c6 (33 aa) 43 GTFGTGGASAQASLASKDKTPKSKSKKGGSTQ [ABP(6G)]c7 (32 aa) 44 KTFKTRKASAQASLASKDKTPKSKSKKGGSTVKNI [ABP(GGspv)] (35 aa) 45 KTFKTRKASAQASLASKDKTPKSKSKKRG [ABP(trc)](29 aa) 46 X₁TFX₂TX₃X₄ASAQASLASKDKTPKSKSKKX₅X₆ [ABP(trc) here X₁ = K, G or A, X₁ = G or A, Consensus (29 X₂ = K, G or V, aa) X₃ = R, G or A, X₄ = K, G or A, X₅ = R, G or V, X₆ = N, G or V, (SEQ ID NO: 46) 47 GTFGTGGASAQASLASKDKTPKSKSKKGG ABP(6G)trc (29 aa) 48 ASEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTC Mouse Fc2a VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQH QDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEE MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY FMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG 49 AEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV Human Fc1x7 VVDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 50 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV Human Fc1X0 VVDVSHEGPEVKFNWHVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPG 51 EVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREF FC5-mFc2a- VSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADY ABP YCAAGSTSTATPLRVDYWGKGTQVTVSSASEPRGPTIKPCPPCKCPAP NLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVN NVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKD LPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPE DIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNS YSCSVVHEGLHNHHTTKSFSRTPGTGGGGSGGGGSKTFKTRKASAQ ASLASKDKTPKSKSKKRNSTQLKSRVKNI 52 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3-mFc2a- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY L-ABP CAAGSTSTATPLRVDYWGQGTLVTVSSASEPRGPTIKPCPPCKCPAP NLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVN NVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKD LPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMP EDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGTGGGGSGGGGSKTFKTRKASAQ ASLASKDKTPKSKSKKRNSTQLKSRVKNI 53 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L-ABP CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSKTFKTRKASAQAS LASKDKTPKSKSKKRNSTQLKSRVKNI 54 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(G) LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSKTFKTRKASAQAS LASKDKTPKSKSKKRGSTQLKSRVKNI 55 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(GG-G) LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSKTFKTG GASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI 56 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(6G) LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSGTFGTGGASAQAS LASKDKTPKSKSKKGGSTQLKSRVKNI 57 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(6G)c1 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSGTFGTGGASAQAS LASKDKTPKSKSKKGGSTQLKSRVK 58 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(6G)c2 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSGTFGTGGASAQAS LASKDKTPKSKSKKGGSTQLKSRV 59 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(6G)c3 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSGTFGTGGASAQAS LASKDKTPKSKSKKGGSTQLKSR 60 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(6G)c4 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSGTFGTGGASAQAS LASKDKTPKSKSKKGGSTQLKS 61 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(6G)c5 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSGTFGTGGASAQAS LASKDKTPKSKSKKGGSTQLK 62 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(6G)c6 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSGTFGTGGASAQAS LASKDKTPKSKSKKGGSTQL 63 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(6G)c7 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSGTFGTGGASAQAS LASKDKTPKSKSKKGGSTQ 64 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(trc) LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD VIAEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSKTFKTRKASAQAS LASKDKTPKSKSKKRG 65 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL ABP(6G)trc LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSGTFGTGGASAQAS LASKDKTPKSKSKKGG 66 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5(H3)-L- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY ABP(GG-G)-L CAAGSTSTATPLRVDYWGQGTLVTVSSGGGGSGGGGSKTFKTGGAS hFc1X7 AQASLASKDKTPKSKSKKRGSTQLKSRVKNIGGGGSGGGGSAEPKSS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 67 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X0-L- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSCDKTHTCPPCPAPEL ABP(GG-G) LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWHVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEGLHNHYTQKSLSLSPGTGGGGSGGGGSKTFKTGGASAQA SLASKDKTPKSKSKKRGSTQLKSRVKNI 68 QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLE IGF1R5-H2- WVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAV hFc1X7-L- YYCAMARQSRVNLDVARYDYWGQGTLVTVSSAEPKSSDKTHTCPPCP ABP(GG-G) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGTGGGGSGGGGSKTFKTGGAS AQASLASKDKTPKSKSKKRGSTQLKSRVKNI 69 KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNIGGGGSGGG ABP(GG-G)-L- GSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT hFc1X7- CVVVDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT IGF1R5-H2 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGQVQL VESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLEWVATI DWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCA MARQSRVNLDVARYDYWGQGTLVTVSS 70 AEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV hFc1X7-L- VVDVSHEGPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL ABP(GG-G)-L- HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE IGF1R5-H2 LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSG GGGSKTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNIGGG GSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQA PGKGLEWVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLR AEDTAVYYCAMARQSRVNLDVARYDYWGQGTLVTVSS 71 EVQLVESGGGLVQPGGSLRLSCAASGFKITHYTMGWFRQAPGKGLEF FC5-H3- VSRITWGGDNTFYSNSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYY hFc1X7- CAAGSTSTATPLRVDYWGQGTLVTVSSAEPKSSDKTHTCPPCPAPEL L(consensus)- LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVKFNWYVDGV ABP(6G) EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGTGX ₁ GX ₂ X ₃ GX ₄ X ₅ GX ₆,GTFGTGGASA QASLASKDKTPKSKSKKGGSTQLKSRVKNI where X₁ = A or G., X₂ = A or G, X₃ = S or T, X₄ = G or V, X₅ = A or G, X₆ = S or T

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1. An isolated peptide that binds β-amyloid comprising an amino acid sequence of: X₁TFX₂TX₃X₄ASAQASLASKDKTPKSKSKKX₅X₆

where X₁=K, G or A or X₁=G or A, X₂=K, G or V, X₃=R, G or A, X₄=K, G or A, X₅=R, G or V, X₆=N, G or V, (SEQ ID NO: 46).
 2. An isolated peptide that binds β-amyloid comprising an amino acid sequence of: (SEQ ID NO: 31) X₁TFX₂TX₃X₄ASAQASLASKDKTPKSKSK KX₅X₆STQLX₇SX₈VX₉NI

wherein X₁=K, G or A, or X₁=G or A, X₂=K, G or V, X₃=R, G or A, X₄=K, G or A, X₅=R, G or V, X₆=N, G or V, X₇=K, G or V, X₈=R, G or A, X₉=K, G or A, or any C-terminally cleaved β-amyloid binding product thereof.
 3. The isolated peptide of claim 1, wherein the amino acid sequence comprises a sequence selected from the group consisting of: (SEQ ID NO: 32) KTFKTRKASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI; (SEQ ID NO: 33) KTFKTRKASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI; (SEQ ID NO: 34) KTFKTRGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI; (SEQ ID NO: 35) KTFKTGGASAQASLASKDKTPKSKSKKRGSTQLKSRVKNI; (SEQ ID NO: 36) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVKNI; (SEQ ID NO: 37) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVK; (SEQ ID NO: 38) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV; (SEQ ID NO: 39) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSR; (SEQ ID NO: 40) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS; (SEQ ID NO: 41) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLK; (SEQ ID NO: 42) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQL; (SEQ ID NO: 43) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQ; (SEQ ID NO: 44) KTFKTRKASAQASLASKDKTPKSKSKKGGSTVKNI; (SEQ ID NO: 45) KTFKTRKASAQASLASKDKTPKSKSKKRG; (SEQ ID NO: 47) GTFGTGGASAQASLASKDKTPKSKSKKGG;

and a sequence that is at least 95% identical to any of the above sequences, or a C-terminally cleaved β-amyloid binding peptide thereof, optionally wherein the C-terminally cleaved β-amyloid binding peptide comprises a sequence selected from the group consisting of: (SEQ ID NO: 37) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRVK; (SEQ ID NO: 38) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSRV; (SEQ ID NO: 39) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKSR; (SEQ ID NO: 40) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLKS; (SEQ ID NO: 41) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQLK; (SEQ ID NO: 42) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQL; and (SEQ ID NO: 43) GTFGTGGASAQASLASKDKTPKSKSKKGGSTQ.


4. (canceled)
 5. The isolated peptide of claim 1, fused to an antibody or antibody fragment capable of transmigrating the blood brain barrier, optionally wherein the antibody or antibody fragment is an Fc fragment.
 6. (canceled)
 7. The isolated peptide of claim 5, wherein the isolated peptide, the antibody or fragment thereof, and the Fc fragment form a single-chain polypeptide fusion protein.
 8. The isolated peptide of claim 5, wherein the Fc fragment comprises an Fc with attenuated effector functions.
 9. A fusion protein comprising the isolated peptide of claim 1, and an antibody or antibody fragment that transmigrates the blood brain barrier (BBB), optionally wherein the fusion protein is a single-chain polypeptide further comprising an Fc fragment, and further optionally wherein the single-chain polypeptide forms a dimer. 10-12. (canceled)
 13. The fusion protein of claim 9, wherein the single-chain polypeptide comprises: an antibody or fragment thereof; a peptide that binds β-amyloid selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47; and an Fc fragment selected from the group consisting of SEQ ID NO: SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, optionally wherein the antibody comprises: a sequence comprising a complementarity determining region (CDR) 1 sequence of GFKITHYTMG (SEQ ID NO:1), a CDR2 sequence of RITWGGDNTFYSNSVKG (SEQ ID NO:2), a CDR3 sequence of GSTSTATPLRVDY (SEQ ID NO:3); a sequence comprising CDR1 sequence of EYPSNFYA (SEQ ID NO:4), a CDR2 sequence of VSRDGLTT (SEQ ID NO:5), a CDR3 sequence of AIVITGVWNKVDVNSRSYHY (SEQ ID NO:6); a sequence comprising CDR1 sequence of GGTVSPTA (SEQ ID NO:7), a CDR2 sequence of ITWSRGTT (SEQ ID NO:8), a CDR3 sequence of AASTFLRILPEESAYTY (SEQ ID NO:9); or a sequence comprising CDR1 sequence of GRTIDNYA (SEQ ID NO:10), a CDR2 sequence of IDWGDGGX: where X is A or T (SEQ ID NO:11), a CDR3 sequence of AMARQSRVNLDVARYDY (SEQ ID NO:12).
 14. (canceled)
 15. The fusion protein of claim 13, wherein the antibody or fragment thereof comprises a sequence selected from any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, and a sequence that is at least 95% identical to any of the above sequences. 16-19. (canceled)
 20. The fusion protein according to claim 13, wherein the single-chain polypeptide comprising the antibody or fragment thereof is linked to the peptide that binds β-amyloid via the Fc fragment, optionally wherein the fusion protein is a single-chain polypeptide comprising a sequences selected from the group consisting of: SEQ ID NO:51 SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO: 54, SEQ ID NO:55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, and a sequence that is at least 95% identical to any of the above sequences, optionally wherein the single-chain polypeptide forms a dimer.
 21. (canceled)
 22. The fusion protein according to claim 20, wherein the fusion protein is a single-chain polypeptide comprising any suitable peptide linker, optionally wherein the peptide linker comprises an amino acid sequence that allows for the linked components of the fusion protein to maintain their unrestricted desired biological function. 23-28. (canceled)
 28. The fusion protein of claim 9, wherein the antibody or fragment thereof is humanized, or a single-domain antibody (sdAb). 29-30. (canceled)
 31. The fusion protein of claim 9, wherein the Fc fragment is mouse Fc2a or human Fc1, optionally wherein the Fc fragment comprises SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO:
 50. 32. The fusion protein of claim 9, wherein the fusion protein comprises the antibody or fragment thereof linked to the N-terminus of the Fc fragment, and the polypeptide that binds β-amyloid linked to the C-terminus of the Fc fragment; or the antibody or fragment thereof linked to the C-terminus of the Fc fragment and the polypeptide that binds β-amyloid linked to the N-terminus of the Fc fragment. 33-37. (canceled)
 38. A pharmaceutical composition comprising the isolated peptide of claim 1, and a pharmacologically acceptable carrier.
 39. A pharmaceutical composition comprising the fusion protein of claim 9, and a pharmacologically acceptable carrier.
 40. A nucleic acid molecule encoding the isolated peptide of claim
 1. 41. A vector comprising the nucleic acid molecule of claim
 40. 42-43. (canceled)
 44. A method of treating Alzheimer's diseases, comprising administering the fusion protein of claim 9 to a subject in need thereof.
 45. A method of reducing β-amyloid levels in a subject having increased levels of β-amyloid comprising the steps of repeated parenteral administration of a sufficient amount of the pharmaceutical composition of claim 39 to a subject. 46-58. (canceled) 